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Structural and functional comparisons between vanadium haloperoxidase and acid phosphatase enzymes
Abstract
The crystallographic structures of both the vanadium chloroperoxidase and bromoperoxidase enzymes have been determined with either vanadium or phosphate bound at their active site. The amino acids that are involved in phosphate binding in the acid phosphatase enzymes and those that are coordinated to vanadium in the haloperoxidases appear to be conserved between the two classes of enzyme. The detailed active site architecture for enzymes that recognize and use either vanadium or phosphate will be discussed in relation to their proposed enzymatic mechanism.
... The group of membrane-bound phosphatases is marked with an M, and the group of soluble phosphatases is marked with an S. [57] The structural similarity of the active sites of V-HPOs and acid phosphatases is also reflected by the fact that phosphate inhibits or inactivates VHPOs and vanadate inhibits the phosphatases. [60][61][62] This similarity has been confirmed by structural determination of an acid phosphatase from Escherichia blattae [63] and its structural comparison with V-CPO (Fig. 6). [61] Striking similarities are also observed from the X-ray structure of the rat prostatic acid phosphatase which has been co-crystallized with a vanadate at its active site. ...
... [60][61][62] This similarity has been confirmed by structural determination of an acid phosphatase from Escherichia blattae [63] and its structural comparison with V-CPO (Fig. 6). [61] Striking similarities are also observed from the X-ray structure of the rat prostatic acid phosphatase which has been co-crystallized with a vanadate at its active site. [64,65] [61,[68][69][70][71][72] Thus, at this time the analogy between these classes of enzymes includes both structural and catalytic aspects. ...
... [61] Striking similarities are also observed from the X-ray structure of the rat prostatic acid phosphatase which has been co-crystallized with a vanadate at its active site. [64,65] [61,[68][69][70][71][72] Thus, at this time the analogy between these classes of enzymes includes both structural and catalytic aspects. However, the detailed structural requirement for these two reactions is still slightly different. ...
... Structural similarities also exist between the VHPOs and phosphatases [224][225][226][227][228][229][230]. The amino acids that are involved in phosphate binding in the acid phosphatase enzymes and also those that are coordinated to vanadium in the VHPOs are also conserved [201,231]. Vanadate(V) as the active site species of VHPOs is covalently bound to a His residue, but the reactivity is in fact determined by the existence of an extensive hydrogen-bonding network. In the case of e.g. the rat prostatic acid phosphatase, the motif is very similar and His12 is the residue bound to vanadate(V), as discussed in Section 4.1 [232]. ...
... Phosphatase activity may indeed be exhibited by apohaloperoxidases [5] and the possibility that peroxidase activity could be observed for vanadate(V)-inhibited phosphatases has also been investigated for several systems [224,226,227,231,233,234]. The first observation of chloroperoxidase activity was reported with phytase, upon addition of vanadate(V) and H 2 O 2 [5,233], and there were several other reports demonstrating that other phosphatases can act as peroxidases in the presence of V V and H 2 O 2 [224,226,227,231,233,234]. ...
... Phosphatase activity may indeed be exhibited by apohaloperoxidases [5] and the possibility that peroxidase activity could be observed for vanadate(V)-inhibited phosphatases has also been investigated for several systems [224,226,227,231,233,234]. The first observation of chloroperoxidase activity was reported with phytase, upon addition of vanadate(V) and H 2 O 2 [5,233], and there were several other reports demonstrating that other phosphatases can act as peroxidases in the presence of V V and H 2 O 2 [224,226,227,231,233,234]. ...
Vanadium is an element ubiquitously present in our planet's crust and thus there are several organisms that use vanadium for activity or function of proteins. Examples are the vanadium-dependent haloperoxidases and the vanadium-containing nitrogenases. Some organisms that use vanadium have extremely efficient and selective protein-dependent systems for uptake and transport of vanadium and are able to accumulate high levels of vanadium from seawater, vanabins being a unique family of vanadium binding proteins found in ascidians involved in this process. For all of the systems a discussion regarding the role of the V-containing proteins is provided, mostly centered on structural aspects of the vanadium site and, when possible or relevant, relating this to the mechanisms operating. Phosphate is very important in biological systems and is involved in an extensive number of biological recognition and bio-catalytic systems. Vanadate(V) is able to inhibit many of the enzymes involved in these processes, such as ATPases, phosphatases, ribonucleases, phosphodiesterases, phosphoglucomutase and glucose-6-phosphatase, and it appears clear that this is closely related to the analogous physicochemical properties of vanadate and phosphate. The ability of vanadium to interfere with the metabolic processes involving Ca2+ and Mg2+, connected with its versatility to undergo changes in coordination geometry, allow V to influence the function of a large variety of phosphate-metabolizing enzymes and vanadate(V) salts and compounds have been frequently used either as inhibitors of these enzymes, or as probes to study the mechanisms of their reactions and catalytic cycle. In this review we give an overview of the many examples so far reported, also disclosing that vanadate(IV) may also have an equally efficient inhibiting effect. The prospective application of vanadium compounds as therapeutics has also been an important topic of research. How vanadium may be transported in blood and up-taken by cells are particularly relevant issues, this being mainly dependent on transferrin (and albumin) present in blood plasma. The thousands of studies reported on the effects of vanadium compounds reflect the complexity of the interactions occurring. Although it is not easy to anticipate/determine if a particular effect observed in a test tube or in vitro is also going to take place in vivo, it is clear that vanadium ions may interfere with many metabolic processes at many distinct levels. Emphasis is given on structural and functional aspects of vanadium-protein interactions relevant for vanadium binding and/or for clarification of role of the metal center in the reaction mechanisms. The additional knowledge that the presence of vanadium can change the action of a protein, other than simply inhibiting it, may also be important to understand how vanadium affects biological systems. This possibility, together with the vanadate-phosphate analogy further potentiates the belief that vanadium probably has relevant functions in living beings, which may involve interaction or incorporation of the metal ion and/or its compounds with several proteins.
... such as wounding, pathogenic attack and light generate reactive oxygen species and 96 H 2 O 2: The vanadium bromoperoxidase will convert this rapidly into HOBr. 97 HOBr has a direct antimicrobial and virucidal effect [32,33] and as shown [26] 98 the oxidized halogen species produced by the marine alga L. digitata deactivate 99 homoserine lactones. These lactones are important communication signals between 100 bacteria and as a result of deactivation biofilm formation and fouling by bacteria and 101 fungi of the surface of the seaweeds is prevented. ...
... The enzymes 497 from the red seaweeds crystallize as dodecamers and the structures are made up 498 of 6 homo-dimers. The subunit in the bromoperoxidases from Cor. officinalis [98] 499 measures approximately 85 56 55Å in size. Twelve subunits are arranged within 500 a 23 cubic point group symmetry. ...
... It is possible to superimpose the secondary structure of the vanadium 506 chloroperoxidase [99] from the fungus Curvularia inaequalis on the Corallina 507 bromoperoxidase dimer. Many of the a-helices of each chloroperoxidase domain 508 are structurally equivalent[98] to the a-helices in the Corallina bromoperoxidase 509 dimer.510 5.8 Active Site of Vanadium Bromoperoxidase 511 from A. Nodosum and Chloroperoxidase 512 from Curvularia inaqualis 513 ...
In this article, the role of vanadium in a number of biological systems will be discussed. The metal oxide (vanadate) is present as the prosthetic group in the haloperoxidases, enzymes that are able to oxidize halides in the presence of hydrogen peroxide to hypohalous acids. The bromoperoxidases seem to be involved in the production of the huge amounts of brominated compounds formed by several seaweeds. The enzymes have unusual properties in terms of stability and structural organization. X-ray structures at high resolution are available now for four vanadium haloperoxidases and details of the events occurring at the active site during catalysis are known. The metal oxide binds peroxide in a side-on fashion and seems to acts as a Lewis acid in polarizing the bound peroxide for further nucleophilic attack by an incoming halide. These enzymes are also able to mediate in the presence of hydrogen peroxide, the enantioselective sulfoxidation of organic sulfides yielding chiral sulfoxides with high enantiomeric excess. The active site of the haloperoxidases is very similar to that found in acid phosphatases that hydrolyze phosphate monoesters and there is some evidence that the haloperoxidases have evolved from the phosphatases. Vanadium is found in the vanadiumIII state in certain blood cells of tunicates (Ascidians) under very acidic conditions. Despite many studies, there is no clue as to the physiological role of the metal and there are many questions regarding the mechanism of accumulation and reduction of the metal in these organisms. The metal also seems to be used as an electron sink by certain bacteria under anaerobic conditions. Furthermore, vanadium is accumulated in Amanita species. These toadstools contain up to 10 mM of a vanadiumIV compound with very special structure in which the vanadiumIV is eightfold coordinated in an unusual geometry. Although the compound has been reported to carry out redox reactions, the physiological function is elusive.
... En revanche, l'enzyme fongique vCPO ne contient qu'un seul site de fixation du vanadium. Le second site aurait été perdu et la région où il aurait dû être présent est maintenant à la surface de l'enzyme (Littlechild et al., 2002). ...
... De plus, ces hélices du site catalytique des vBPO sont quasiment superposables à celles du fagot 1. Cette observation a servi de base à l'hypothèse qu'une duplication du gène ancestral, similaire aux vBPO algales, aurait eu lieu et abouti chez le champignon à une structure monomérique qui possède les deux fagots d'hélices au sein de la même chaine. Un des sites actifs aurait été perdu par la suite (Littlechild et al., 2002). ...
The surface of algae is colonized by marine heterotrophic bacteria which entertain different trophic relationships with their host. These relationships may be of various types such as symbiosis, commensalism, saprophytic or pathogenic. In all cases the algal cell wall, which is mainly constituted by polysaccharides, is the first barrier bacteria have to interact with. Therefore, bacteria have developed sophisticated machineries (involving various glycoside hydrolases) to use the cell wall components as carbon source, but they also have to cope with algal defence reactions, which are also located in the cell wall of algae. Zobellia galactanivorans is a marine flavobacteria whose genome has recently been sequenced. It represents a model for the interaction between algae and bacteria. Indeed, 3 % of genes are involved in polysaccharide degradation, many of them organised in operon-like structures. Moreover, many enzymes present in the genome appear to be involved in original metabolisms, such as iodine utilization. My interest focused on the molecular aspects of three different factors involved in interaction of Z. galactanivorans with algae. The genome of Z. galactanivorans revealed two new sequences of glycosides hydrolases family 82 which are particular, due to their truncated sequences. Based on multiple sequence alignments, I designed site directed mutagenesis experiments, performed with the Alteromonas fortis enzyme, the first structural representant of iota-carrageenases. The results show that E245 (proton donor) and D247 (nucleophile) are the catalytic residues and that residues Q222, H281 and E310 also play crucial roles in the enzymatic reaction. Using a crystallographic approach, I was able to highlight the importance of a chloride ion in the formation of a water network, close to the active site. I also initiated the first biochemical and structural characterization of two members of a new GH family, located in operon-like gene organisations apparently involved in the degradation of sulphated polysaccharides. A third part of my thesis concerns the crystallographic structure determination of a first prokaryote, vanadium dependent iodoperoxydase, identified in the Z. galactanivorans genome. The structural analysis of this enzyme that may be involved in a detoxification reaction following the oxidative burst generated by algae, allows us to suggest a new evolution pattern for this type of enzymes.
... Comparison of the crystal structures of VBPOs and VCPOs reveals an unexpected homology that the vanadate-binding residues largely overlap ( Figure 4) despite the fact that these enzymes are distinct from each other. 15,91 However, the primary amino acid sequences are conserved to only $20% across the various families of VHPOs. 69 Moreover, there are a few residues near the active site that differ between VCPOs and VBPOs such as Arg-395 in VBPOs is a tryptophan in VCPO, and His-480 in VBPOs is a phenylalanine in VCPOs, while His-411 residue in VBPO (from A. nodosum) is substituted by Phe-397 in VCPO ( Figure 4). ...
... Whether the substrate reacts predominantly with free HOX or with some form of enzymebound V-OX (X¼ halide) intermediate is still uncertain. 15,91 Some results dictate that these enzymes can catalyze reactions with regioselectivity or enantioselectivity, demonstrating the involvement of trapped X þ equivalents 15,108,109 ; whereas others show no selectivity or mechanistic flexibility. 15 Anderson et al. discovered that the VBPO isolated from the alga Corallina officinalis catalyzes the oxidation of bicyclic sulfides to the corresponding sulfoxides with 91% enantiomeric excess. ...
Vanadium is a trace element and exists in biological systems, both as a required element and as a mimic of other required metal ions or phosphorus. Several classes of enzymes contain vanadium in the active site, including vanadium-dependent haloperoxidases, vanadium-containing nitrogenases, and vanadium-binding proteins, vanabins. Haloperoxidases are well characterized because in addition to the mechanism by which they oxidize substrates they are also important in biotechnology and are used as oxidation catalysts. The vanadium-containing cofactor for nitrogenases has been characterized, as well as the vanabins. The mushrooms of the genus Amanita muscaria store vanadium as the only known vanadium-containing natural product amavadine, which is a non-oxovanadium(IV) compound with unique coordination chemistry. Several ascidians and fan worms accumulate vanadium mainly in its lower oxidation state +. 3, and the role of the vanadium and the mechanism for bioprocessing are poorly understood. In addition to vanadium being a required component of proteins, it has been reported to have biological properties. For example, vanadium compounds have been shown to induce an insulin-enhancing response in diabetic animal model systems and human beings. This chapter summarizes these areas of bioinorganic chemistry relating to vanadium.
... V-HPOs containing a ligated vanadate ion utilize hydrogen peroxide to oxidize halide ions to the corresponding hypohalous acid. Interestingly, acid phosphatases have very similar active site architecture to that of VHPOs and are able to utilize vanadate as well as phosphate [2,3]. Several structures of haloperoxidases have been solved, either with a vanadate ion or a phosphate ion as a ligand. ...
... It has been shown that the amino acids in the vanadium-binding site clefts of vanadium dependent haloperoxidases and acid phosphatases are highly conserved [2,3]. The sequence homology and related phosphatase activities suggest evolutionary relationships between the groups of enzymes belonging to three families of acid phosphatases. ...
The rapidly growing number of protein structures in the protein data bank (PDB) provides opportunities to obtain biological insights from those that share common features. We used the information available in the PDB to survey the structural features that are common among proteins, which bind to vanadate. The ability of vanadate to mimic phosphate and vice versa is presumably due to the structural similarity vanadate shares with phosphate. We analyze the geometries of the bound vanadate and the nature of its binding interfaces. We also compared structures of enzyme groups bound to both ligands.
This data improves our understanding of vanadate recognition and will be useful in not only the evaluation of new structures but also in the development of new therapeutic agents based on structural recognition.
... The dephosphorylation of phosphatidate is catalyzed by PAP2 enzymes and produces diacylglycerol with inorganic phosphate. In eukaryotic cells, especially in the synthesis of phospholipids and triacylglycerol, PAP plays a key role through its product diacylglycerol, and it also produces and/or degrades lipidsignalling molecules that are related to phosphatidate (Littlechild et al., 2002). ...
Francisella tularensis Schu S4 is the causal agent of a sporadic zoonotic disease known as Tularemia, which has shown epidemic outbreaks recently in certain parts of the world. This pathogen is a potential agent of biowarfare or bioterrorism and is classified as a category A pathogen by the National Institute of Allergy and Infectious Diseases. In this virulent strain, 453 genes have been identified as essential genes, indispensable for growth and survival of the pathogen. The functions of 44 proteins encoded by those essential genes were found to be hypothetical and thus defined as essential hypothetical proteins (EHPs). The current study used a wide range of in silico tools and servers to annotate the physicochemical, structural, and functional properties of these EHPs. Of all the EHPs, 24 were functionally annotated with a high degree of confidence and validated by Receiver Operating Characteristic curve analysis. Non-homology assessment revealed 20 pathogen-specific EHPs, which were further analyzed for protein-protein interactions and predicted for secondary and tertiary structure. All the 3D structures were checked on multiple quality assessment servers, and the best models were visualized. The outcome of the study could aid in enhancing current understanding of bacterial pathogenesis with novel drug and vaccine investigations.
... The dephosphorylation of phosphatidate is catalyzed by PAP2 enzymes and produces diacylglycerol with inorganic phosphate. In eukaryotic cells, especially in the synthesis of phospholipids and triacylglycerol, PAP plays a key role through its product diacylglycerol, and it also produces and/or degrades lipidsignalling molecules that are related to phosphatidate (Littlechild et al., 2002). ...
Francisella tularensis Schu S4 is the causal agent of a sporadic zoonotic disease known as Tularemia, which has shown epidemic outbreaks recently in certain parts of the world. This pathogen is a potential agent of biowarfare or bioterrorism and is classified as a category A pathogen by the National Institute of Allergy and Infectious Diseases. In this virulent strain, 453 genes have been identified as essential genes, indispensable for growth and survival of the pathogen. The functions of 44 proteins encoded by those essential genes were found to be hypothetical and thus defined as essential hypothetical proteins (EHPs). The current study used a wide range of in silico tools and servers to annotate the physicochemical, structural, and functional properties of these EHPs. Of all the EHPs, 24 were functionally annotated with a high degree of confidence and validated by Receiver Operating Characteristic curve analysis. Non-homology assessment revealed 20 pathogen-specific EHPs, which were further analyzed for protein-protein interactions and predicted for secondary and tertiary structure. All the 3D structures were checked on multiple quality assessment servers, and the best models were visualized. The outcome of the study could aid in enhancing current understanding of bacterial pathogenesis with novel drug and vaccine investigations.
... Conversely, phosphate inhibits vanadate reduction in Shewanella oneidensis (Carpentier et al., 2003). Sequence alignment and crystal structure analysis of vanadium haloperoxidases has revealed that amino acids that bind the vanadate cofactor are conserved with a family of acid phosphatases (Hemrika, Renirie, Dekker, Barnett, & Wever, 1997;Littlechild, Garcia-Rodriguez, Dalby, & Isupov, 2002;Wever & Hemrika, 2001). Recently, it has been shown that an increase in arsenate (AsO ...
The incorporation of metal cofactors into protein active sites and/or active regions expanded the network of microbial metabolism during the Archean eon. The bioavailability of crucial metal cofactors is largely influenced by earth surface redox state, which impacted the timing of metabolic evolution. Vanadium (V) is a unique element in geo-bio-coevolution due to its complex redox chemistry and specific biological functions. Thus, the extent of microbial V utilization potentially represents an important link between the geo- and biospheres in deep time. In this study, we used geochemical modeling and network analysis to investigate the availability and chemical speciation of V in the environment, and the emergence and changing chemistry of V-containing minerals throughout earth history. The redox state of V shifted from a more reduced V(III) state in Archean aqueous geochemistry and mineralogy to more oxidized V(IV) and V(V) states in the Proterozoic and Phanerozoic. The weathering of vanadium sulfides, vanadium alkali metal minerals, and vanadium alkaline earth metal minerals were potential sources of V to the environment and microbial utilization. Community detection analysis of the expanding V mineral network indicates tectonic and redox influence on the distribution of V mineral-forming elements. In reducing environments, energetic drivers existed for V to potentially be involved in early nitrogen fixation, while in oxidizing environments vanadate (
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) could have acted as a metabolic electron acceptor and phosphate mimicking enzyme inhibitor. The coevolving chemical speciation and biological functions of V due to earth's changing surface redox conditions demonstrate the crucial links between the geosphere and biosphere in the evolution of metabolic electron transfer pathways and biogeochemical cycles from the Archean to Phanerozoic.
... (Source: Wikipedia). major function of V-HPOs is to incorporate halogen atoms into organic molecules (Figure 13) 37,53 . The blood of some species of ascidians and tunicates, also known as sea squirts and sea cucumbers, contains proteins called vanabins where V III presents in much higher concentration (~350 mM) 54 . ...
... Alternatively, PhoD and PhoX enzymes both require Fe 3þ and Ca 2þ as cofactors Yong et al., 2014) and βPPhy also requires Ca 2þ (Mullaney and Ullah, 2003). The other proteins all require different co-factors; Zn 2þ and Mg 2þ for PhoA (Torraini, 1990), VO 4 3À for the NSAPs (Littelchild et al., 2002). The acid phytases CPhy and HAPhy have no known requirement for metal cofactors. ...
The mechanisms by which microbial communities maintain functions within the context of changing environments are key to a wide variety of environmental processes. In soil, these mechanisms support fertility. Genes associated with hydrolysis of organic phosphoesters represent an interesting set of genes with which to study maintenance of function in microbiomes. Here, we shown that the richness of ecotypes for each gene varies considerably in response to application of manure and various inorganic fertilizer combinations. We show, at unprecedented phylogenetic resolution, that phylogenetic diversity of phosphohydrolase genes are more responsive to soil management and edaphic factors than the taxonomic biomarker 16S rRNA gene. Available phosphorus – assessed by measuring Olsen-P - exerted some influence on alkaline phosphatase distribution: however, consistent and significant differences were observed in gene abundance between treatments that were inconsistent with bioavailable orthophosphate being the dominant factor determining gene abundance. Instead, we observed gene niche separation which was most strongly associated with soil exchangeable calcium. Our study suggests that the bioavailability of enzyme cofactors (exchangeable calcium in the case of phoD, phoX and βPPhy studied here) influence the abundance of genes in soil microbial communities; in the absence of cofactors, genes coding for alternative enzyme families that do not require the limiting cofactor (for example, non-specific acid phosphatases which require vanadate) become more abundant.
... Besides, it showed 83% identity with the well-characterized chloroperidase from Streptomyces lividans (Bantleon et al., 1994). Mechanisms, functionality and structure of chloroperoxidases were previously studied (Littlechild et al., 2002;Timmins and de Visser, 2015). Based on this background, this enzyme may serve as a detoxification tool for Streptomyces sp. ...
Highly contaminated γ-hexachlorocyclohexane (lindane) areas were reported worldwide. Low aqueous solubility and high hydrophobicity make lindane particularly resistant to microbial degradation. Physiological and genetic Streptomyces features make this genus more appropriate for bioremediation compared with others. Complete degradation of lindane was only proposed in the genus Sphingobium although the metabolic context of the degradation was not considered. Streptomyces sp.M7 has demonstrated ability to remove lindane from culture media and soils. In this study, we used MS-based label-free quantitative proteomic, RT-qPCR and exhaustive bioinformatic analysis to understand lindane degradation and its metabolic context in Streptomyces sp. M7. We identified the proteins involved in the up-stream degradation pathway. In addition, results demonstrated that mineralization of lindane is feasible since proteins from an unusual down-stream degradation pathway were also identified. Degradative steps were supported by an active catabolism that supplied energy and reducing equivalents in the form of NADPH. To our knowledge, this is the first study in which degradation steps of an organochlorine compound and metabolic context are elucidate in a biotechnological genus as Streptomyces. These results serve as basement to study other degradative actinobacteria and to improve the degradation processes of Streptomyces sp. M7.
... 50,87,88 Structurally, the vanadium-dependent haloperoxidases are similar to the acid phosphatases; therefore, it is not surprising that these enzymes can bind phosphate in place of vanadate and exhibit phosphatase activity. 89 Phosphate buffers can be therefore useful for crystallization of the enzymes, but should be avoided in assays as phosphate can act as a competitive inhibitor. 90,91 The vanadate ion is bound at the bottom of a wide funnel, which is between 15 and 20 Å in length at the core of two four helix bundles. ...
Nature has evolved halogenase enzymes to regioselectively halogenate a diverse range of biosynthetic precursors, with the halogens introduced often having a profound effect on the biological activity of the resulting natural products. Synthetic endeavors to create non-natural bioactive small molecules for pharmaceutical and agrochemical applications have also arrived at a similar conclusion: halogens can dramatically improve the properties of organic molecules for selective modulation of biological targets in vivo. Consequently, a high proportion of pharmaceuticals and agrochemicals on the market today possess halogens. Halogenated organic compounds are also common intermediates in synthesis and are particularly valuable in metal-catalyzed cross-coupling reactions. Despite the potential utility of organohalogens, traditional nonenzymatic halogenation chemistry utilizes deleterious reagents and often lacks regiocontrol. Reliable, facile, and cleaner methods for the regioselective halogenation of organic compounds are therefore essential in the development of economical and environmentally friendly industrial processes. A potential avenue toward such methods is the use of halogenase enzymes, responsible for the biosynthesis of halogenated natural products, as biocatalysts. This Review will discuss advances in developing halogenases for biocatalysis, potential untapped sources of such biocatalysts and how further optimization of these enzymes is required to achieve the goal of industrial scale biohalogenation.
... Structural and functional comparisons of V-HPOs from eukaryotic organisms have shown that seven amino acids are required for halogenating activity. 217,[223][224][225] All three nap V-HPO enzymes contain His-496 (which covalently binds to the vanadate cofactor) and five of the six residues that participate in the hydrogen bonding of vanadate: Lys-353, Arg-360, Ser-402, Gly-403, and Arg-490 (numbering from C. inaequalis). Conserved residue His-404, which is proposed to form a hydrogen bond to the apical oxygen of the cofactor, is replaced with Ser, as seen in NapH1 and NapH4, or Phe as seen in NapH3. ...
Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of nature's halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.
... (Fig. 25). 308 Based on these structural and functional similarities, the authors incorporated vanadate ion into phosphatases to produce a novel, semi-synthetic peroxidases. The resulting vanadium phosphatase catalyzed the enantioselective oxidation of several sulfides, with slight ee. ...
Many efforts are being made in the design and engineering of metalloenzymes with catalytic properties fulfilling the needs of practical applications. Progress in this field has recently been accelerated by advances in computational, molecular and structural biology. This review article focuses on the recent examples of oxygen-activating metalloenzymes, developed through the strategies of de novo design, miniaturization processes and protein redesign. Considerable progress in these diverse design approaches has produced many metal-containing biocatalysts able to adopt the functions of native enzymes or even novel functions beyond those found in Nature.
... During the course of divergent evolution, the bacterial V-HPO enzymes may become clustered independently and have similarities to the type 2 phosphatidic acid phosphatase (PAP2) family [55]. The PAP2 superfamily includes the bacterial non-specific acid phosphatases and a variety of HPO enzymes, which may share a similar evolutionary history [12,56]. Structure-based MSA analysis showed that the V-HPO enzymes contain many α-helices, and the C-terminal has several conserved sites (S5 Fig). ...
Halogenated natural products are widespread in the environment, and the halogen atoms are typically vital to their bioactivities. Thus far, six families of halogenating enzymes have been identified: cofactor-free haloperoxidases (HPO), vanadium-dependent haloperoxidases (V-HPO), heme iron-dependent haloperoxidases (HI-HPO), non-heme iron-dependent halogenases (NI-HG), flavin-dependent halogenases (F-HG), and S-adenosyl-L-methionine (SAM)-dependent halogenases (S-HG). However, these halogenating enzymes with similar biological functions but distinct structures might have evolved independently. Phylogenetic and structural analyses suggest that the HPO, V-HPO, HI-HPO, NI-HG, F-HG, and S-HG enzyme families may have evolutionary relationships to the α/β hydrolases, acid phosphatases, peroxidases, chemotaxis phosphatases, oxidoreductases, and SAM hydroxide adenosyltransferases, respectively. These halogenating enzymes have established sequence homology, structural conservation, and mechanistic features within each family. Understanding the distinct evolutionary history of these halogenating enzymes will provide further insights into the study of their catalytic mechanisms and halogenation specificity.
... This similarity has been confirmed by the structural determination of an acid phosphatase from Escherichia blattae (108). Acid phosphatases that are related to the VHPOs enzymes are considered to be histidine phosphatases (108,109 The kinetics and mechanism of haloperoxidases have been the subject of multiple studies (110,111) in part due to their potential use as industrial antimicrobial agents and disinfectants (112,113) as well as halogenation catalysts (114,115). A few thermostability studies have also been done (e.g. ...
... In VBPO dimers, the surface of interaction represents 20 and 46% of the total exposed surface of a monomer for C. officinalis [93] and A. nodosum [39], respectively. In contrast, in the red algal VBPO dodecamer, only 12% of a monomer surface is exposed to the solvent [93,96,97]. ...
In the environment, vanadium-dependent haloperoxidases (VHPO) are likely to play a key role in the production of biogenic organo-halogens. These enzymes contain vanadate as a prosthetic group, and catalyze, in the presence of hydrogen peroxide, the oxidation of halide ions (Cl−, Br− or I−). They are classified according to the most electronegative halide that they can oxidize. Since the first discovery of a vanadium bromoperoxidase in the brown alga Ascophyllum nodosum thirty years ago, structural and mechanistic studies have been mainly conducted on two types of VHPO, chloro- and bromoperoxidases, and more recently on a vanadium-dependent iodoperoxidase. In this review, we highlight the main progress obtained on the structure-function relation of these proteins, based on biochemistry, crystallography and X-ray absorption spectroscopy (XAS). The comparison of 3D protein structures of the different VHPO helped identify the residues that govern the molecular mechanisms of catalysis and specificity of VHPO. Vanadium K-edge XAS gave further important insight to understand the fine changes around the vanadium cofactor during the catalytic cycle. The combination of different structural approaches, at different scales of resolution, shed new light on biological vanadium coordination in the active site, and its importance for the catalytic cycle and halide specificity of vanadium haloperoxidases.
... Vanadate and vanadate derivatives have been employed to interrogate a range of enzymes that interact with phosphorylated substrates (31). Acid phosphatase enzymes have evolved to accommodate vanadate as a redox cofactor (32,33). ...
Introduction:
Bacterial nonspecific acid phosphohydrolases (NSAPs) or phosphatases are group of enzymes secreted as soluble periplasmic proteins or retained as membrane bound lipoproteins that are usually able to dephosphorylate a broad array of structurally unrelated organic phosphoesters (nucleotides, sugar phosphates, phytic acid etc.) to acquire inorganic phosphate (Pi) and organic byproducts. They exhibit optimal catalytic activity at acidic to neutral pH values. On the basis of amino acid sequence relatedness, phosphatase are grouped into different molecular families namely Class A, Class B and Class C acid phosphatase respectively.
Results and discussion:
In this article out of thirty three sequences, twenty six belonging to each of the three classes of bacterial acid phosphatase and seven belonging to archaeal phosphoesterases were analyzed using various tools of bioinformatics. Phylogenetic analysis, dot plot comparisons and motif analysis were done to identify a number of similarities and differences between three classes of bacterial acid phosphatases and archaeal phosphoesterases. In this research we have attempted to decipher evolutionary relationship between three classes of bacterial acid phosphatase and archaeal phosphoesterases using bioinformatics approach.
... In brown algae, vanadium is used both extracellularly in its available form (V V ) as the cofactor of apoplastic vanadium-dependant haloperoxidases (V-HPO), thereby eluding toxicity problems, and intracellularly probably via appropriate chelators, as cofactor of cytoplasmic haloperoxidases. Algal V-HPOs are redox-inert (V V ) with a catalytic site featuring a coordinate chemistry identical to that of acid phosphatases (Hemricka et al., 1997, Littlechild et al., 2002). Indeed, the similarity between vanadate and phosphate is the favoured explanation for vanadate accumulation in vertebrate bones, and hence for the vanadate complex as non-heme cofactor of peroxidases, in lieu of phosphate. ...
Marine biodiversity as we see it today on our planet is the result of some four billion years of evolution of life. If the initial stages of this story are conjectural, all life forms have always been associated with the presence of water and its associated chemistries. With metabolisms adapting to forms of energy progressively made available, with the conquest of new media and new territories, biodiversity has made quantum jumps and suffered occasional natural catastrophes. Under stabilized climatic eras, marine communities generate around natural architectures, sometimes of colossal dimensions, such as kelp beds or coral reefs. Complex niches appear in order to accommodate life forms having different trophic requirements, some life forms serving as food or as substrate or as carrier to other life forms. Chemical diversity is well reflected by the sophistication of the metabolic repertoire and its associated transcriptomic syntax, in a molecular language necessarily elaborate to support biodiversity on a sustainable basis. Yet today this stability can be harmed by human overcontrol of the environment, for example by producing a vast number of new chemicals which compete with natural molecules in an unfavorable way. Finally, chemical elements or molecules which have a special role in the marine world will be given as examples of 'evolutionary singularities'.
The understanding of the role of vanadium enzymes and of vanadium compounds (VCs) in biology, as well as the design of new vanadium-based species for catalysis, materials science and medicinal chemistry has exponentially increased during the last decades. In biological systems, VCs may rapidly interconvert under physiological conditions and several V-containing moieties may be formed and bind to proteins. These interactions play key roles in the form transported in blood, in the uptake by cells, in inhibition properties and mechanism of action of essential and pharmacologically active V species. In this review, we focus on the recent advances made, namely in the application of the theoretical methodologies that allowed the description of the coordinative and non-covalent VC–protein interactions. The text is organized in six main topics: a general overview of the most important experimental and computational techniques useful to study these systems, a discussion on the nature of binding process, the recent advances on the comprehension of the V-containing natural and artificial enzymes, the interaction of mononuclear VCs with blood and other physiologically relevant proteins, the binding of polyoxidovanadates(V) to proteins and, finally, the biological and therapeutic implications of the interaction of pharmacologically relevant VCs with proteins and enzymes. Recent developments on vanadium-containing nitrogenases, haloperoxidases and nitrate reductases, and binding of VCs to transferrin, albumins, immunoglobulins, hemoglobin, lysozyme, myoglobin, ubiquitin and cytochrome c are discussed. Challenges and ideas about desirable features and potential drawbacks of VCs in biology and medicine and future directions to explore this chemistry area are also presented. The deeper understanding of the interactions of V-species with proteins, and the discussed data may provide the basis to undertake the investigation, design and development of new potentially active VCs with a more solid knowledge to predict their binding to biological receptors at a molecular point of view.
Vanadium haloperoxidases are one of the few enzymes in nature that utilize a vanadium center and catalyze the halogenation of substrates through the biosynthesis of hypohalite. Vanadium chloroperoxidases (VCPOs) bind and activate hydrogen peroxide and in a reaction with chloride convert it into hypochlorite as a precursor for a substrate chlorination reaction. Despite the fact that these enzymes have been studied extensively, surprisingly little is known on their catalytic cycle and particularly on the function of the vanadium atom in the reaction mechanism. In order to gain insight into the intricate details of the catalytic cycle of VCPOs, we performed an extensive computational study using large cluster model complexes, where we tested many possible pathways and active-site protonation states. Our work establishes that the biosynthesis of hypochlorite proceeds in two steps: H2O2 activation on the vanadium center to form an end-on V(V)-hydroperoxo complex, followed by OH+ transfer from hydroperoxo to chloride on the vanadium center to form hypochlorite. We show that the initial reaction starts with a proton transfer from H2O2 to the equatorial OH group of the VV(O)2(OH)2- active site, followed by hydroperoxo binding and water release to form the highly stable vanadium-hydroperoxo-dioxo-hydroxo complex. A further proton transfer from an active-site His or Lys residue can lead to the vanadium-peroxo-hydroxo-oxo complex, which we assign as a dead-end complex unable to react further to hypochlorite products. The mechanisms were considered under various protonation state, and it is shown to be the most effective with His404 singly protonated. The work shows that vanadium is a spectator ion that does not change its oxidation state during the reaction mechanism but holds and positions the H2O2 substrate and guides its proton-relay steps through its oxo and hydroxo ligands. The effect of the protonation state of first- and second-coordination sphere residues and ligands was tested and shows that the reaction is highly sensitive to local changes in the protonation state. Finally, the computations show that the oxygen atom of HOCl exclusively derives from H2O2.
Vanadium is one of the more abundant elements in the Earth’s crust and exhibits a wide range of oxidation states in its compounds making it potentially a more sustainable and more economical choice as a catalyst than the noble metals. A wide variety of reactions have been found to be catalysed by homogeneous, supported and heterogeneous vanadium complexes and the number of applications is growing fast.
Bringing together the research on the catalytic uses of this element into one essential resource, including theoretical perspectives on proposed mechanisms for vanadium catalysis and an overview of its relevance in biological processes, this book is a useful reference for industrial and academic chemists alike.
Recent work revealed an active biological chlorine cycle in coastal Arctic tundra of northern Alaska. This raised the question whether chlorine cycling was restricted to coastal areas, or if these processes extended to inland tundra. The anaerobic process of organohalide respiration, carried out by specialized bacteria like Dehalococcoides, consumes hydrogen gas and acetate using halogenated organic compounds as terminal electron acceptors, potentially competing with methanogens that produce the greenhouse gas, methane. We measured microbial community composition and soil chemistry along a ~262 km coastal-inland transect to test for the potential of organohalide respiration across the Arctic Coastal Plain, and studied the microbial community associated with Dehalococcoides to explore the ecology of this group and its potential to impact C cycling in the Arctic. Brominated organic compounds declined sharply with distance from the coast, but decrease in organic chlorine pools was more subtle. The relative abundance of Dehalococcoides was similar across the transect, except being lower at the most inland site. Dehalococcoides correlated with other strictly anaerobic genera, plus some facultative ones, that had the genetic potential to provide essential resources (hydrogen, acetate, corrinoids, or organic chlorine). This community included iron reducers, sulfate reducers, syntrophic bacteria, acetogens and methanogens, some of which might also compete with Dehalococcoides for hydrogen and acetate. Throughout the Arctic Coastal Plain, Dehalococcoides is associated with the dominant anaerobes that control fluxes of hydrogen, acetate, methane and carbon dioxide. Depending on seasonal electron acceptor availability, organohalide respiring bacteria could impact carbon cycling in Arctic wet tundra soils.
Importance
Once considered relevant only in contaminated sites, it is now recognized that biological chlorine cycling is widespread in natural environments. However, linkages between chlorine cycling and other ecosystem processes are not well established. Species in the genus Dehalococcoides are highly specialized, using hydrogen, acetate, vitamin B12-like compounds and organic chlorine produced by the surrounding community. We studied which neighbors might produce these essential resources for Dehalococcoides species. We found that Dehalococcoides are ubiquitous across the Arctic Coastal Plain and are closely associated with a network of microbes that produce or consume hydrogen or acetate, including the most abundant anaerobic bacteria and methanogenic archaea. We also found organic chlorine and microbes that can produce these compounds throughout the study area. Therefore, Dehalococcoides could control the balance between carbon dioxide and methane (a more potent greenhouse gas) when suitable organic chlorine compounds are available to drive hydrogen and acetate uptake.
Vanadium-oxo and vanadium-peroxo complexes are common intermediates in biology and are, for instance, found in the catalytic cycle of vanadium haloperoxidases. In biomimetic chemistry synthetic models have been created that mimic the structural features of the coordination environment of these vanadium-oxo and vanadium-peroxo species. Recently, two novel vanadium-oxo complexes were trapped and characterized with a trigonal bipyramidal ligand design with either a solvent exposed vanadium center or the vanadium inside a cage, designated as the bowl-shaped configuration and the dome-shaped structure, respectively. Density functional theory calculations are reported here on these bowl- and dome-shaped structures where we study the reaction with t-butylhydroperoxide to form the vanadium-peroxo species and its reaction with thioanisole. Although the structural features of the vanadate core are close for both structures, the calculations display a strong second-coordination sphere effect of the ligand architecture on the barrier heights of the reaction with a terminal oxidant even though the rate-determining transition states show little structural differences. A similar observation is seen for the reaction of the two vanadium-peroxo species with thioanisole. Overall, the calculations implicate that vanadium-peroxo is an efficient oxidant of sulfoxidation reactions, although it is not as efficient as analogous iron(IV)-oxo heme and nonheme oxidants that react with substantially lower barriers. The reactivity differences are analyzed with thermochemical cycles and valence bond patterns that explain the differences in chemical properties and identify how the ligands affect the chemical reactivity with substrates.
Vanadium porphyrin complexes are naturally occuring substances found in crude oil and have been shown to have medicinal properties as well. Little is known on their activities with substrates; therefore, we decided to perform a detailed density functional theory study into the properties and reactivities of vanadium(IV)- and vanadium(V)-oxo complexes with a TPPCl8 or 2,3,7,8,12,13,17,18-octachloro-meso-tetraphenylporphyrinato ligand system. In particular, we investigated the reactivity of [VV(O)(TPPCl8)]+ and [VIV(O)(TPPCl8)] with cyclohexene in the presence of H2O2 or HCO4–. The work shows that vanadium(IV)-oxo and vanadium(V)-oxo are sluggish oxidants by themselves and react with olefins slowly. However, in the presence of hydrogen peroxide these metal-oxo species can be transformed into a side-on vanadium-peroxo complex, which reacts with substrates more efficiently. Particularly with anionic axial ligands, the side-on vanadium-peroxo and vanadium-oxo complexes produced epoxides from cyclohexene with small barier heights. In addition to olefin epoxidation, we investigated aliphatic hydroxylation mechanisms of the same oxidants and some oxidants show efficient and viable cyclohexene hydroxylation mechanisms. The work implies that vanadium-oxo and vanadium-peroxo complexes can react with double bonds through epoxidation, and under certain conditions also give hydroxylation, but the overall reactivity is highly dependent on the equatorial ligand, the local environment and the presence of anionic axial ligands.
Vanadium Haloperoxidases (VHPOs) have been used in a variety of biotransformations showing remarkable stereoselectivity and regiospecificity. The high efficiency of the enzyme is influenced by the protein active site and the role of certain amino acids in activation of vanadium(V)-bound peroxide for halide oxidation. The use of natural or recombinant enzymes, or biomimetic vanadium compounds brings up issues regarding the cost of production and reaction conditions. In this chapter, the primary intent is to provide a simple and clear picture of functional mimicking nature of peroxovanadium compounds with haloperoxidases enzymes to the readers. Major emphasis would be given to examine the reactivity of the vanadium haloperoxidases with mechanism.
Many natural compounds are halogenated and the enzymes that carry out these reactions have been characterized. This chapter provides a brief overview of the different types of halogenating enzymes characterized to date and their differing structures. It concentrates specifically on the vanadium haloperoxidases with regard to their structure and mechanism and their uses in industrial biocatalysis. Many new drugs entering the market are halogenated and this modification is known to change their biological activity. The use of the enzymes known to carry out these specific reactions in ‘nature’ is important for the industrial biosynthesis of new drug molecules and halogenated building blocks for the pharmaceutical industry.
This chapter describes the different natural enzymes that have been discovered that can specifically halogenate organic compounds and that have applications in biotransformation reactions. Specific halogenation of a compound can change its chemical and biological properties. There are many naturally halogenated compounds that have been isolated from both marine and terrestrial environments and some examples are presented. Many of these compounds have properties that are of interest to the pharmaceutical industry since they have antibacterial or anticancer activities. This chapter summarizes the enzymes discovered to date and discusses their properties. They include heme haloperoxidases, nonheme iron-dependent halogenases, vanadium haloperoxidases, flavin-dependent haloperoxidases, noncofactor-containing bacterial haloperoxidases, S-adenosyl-. l-methionine-dependent chlorinases and fluorinases, and methyl halide transferases. These enzymes are unrelated structurally and use different mechanisms to achieve their chemical reactions. The known applications of well-studied enzymes are discussed alongside potential applications of more recently discovered enzymes for white biotechnology.
This chapter will discuss the application of enzymes to carry out biotransformation reactions for the synthesis of building blocks of new drugs within the fine chemicals industry. It will concentrate on the marine environment to discover novel enzymes that have applications in this important area of substainable chemistry. Marine enzymes that have been cloned and isolated from bacteria, archaea, macro algae and viruses will be used to illustrate specific examples and applications. These enzyme activities include haloperoxidases, dehalogenases, alcohol dehydrogenases, L-aminoacylases, proteases, esterases and lipases. The biochemical and structural studies on these marine enzymes will be described in relation to their mechanism of action and evolutionary diversity with regards to related enzymes.
Haloperoxidases have been the only halogenating enzymes know for more than 35 years. They produce in general free hypohalous acid which acts as the halogenating agent and leads thus to a product formation almost identical to chemical halogenation reactions using electrophilic halogen species. With the detection of FADH2-dependent halogenases a type of enzymes was found that shows high substrate specificity and catalyzes regioselective halogenation reactions. FADH2-dependent halogenases are involved in many biosynthetic pathways and catalyze the halogenation of aromatic and aliphatic substrates activated for electrophilic attack. These enzymes also produce hypohalous acids. However, in contrast to haloperoxidases, the hypohalous acid generated cannot leave the active site but can only react with substrates at the active site resulting in selective halogenation reactions. FADH2-dependent halogenases are a twocomponent system with a flavin reductase as the second component. They show similarity to flavin-dependent monooxygenases in forming an enzyme-bound flavin hydroperoxide intermediate. This flavin hydroperoxide reacts with a halide to form hypohalous acid which may then react in a selective reaction with the organic substrate. For halogenation of unactivated carbon atoms, non-heme iron, α- ketoglutarate- and O2-dependent halogenases use a radical mechanism. A substrate radical intermediate which is formed by abstraction of a H. abstracts a halide radical from the non-heme iron coordination complex resulting in the formation of a halogenated methyl group. While in vitro application of both types of halogenases is problematic, due to issues with cofactor recycling, in vivo use which overcomes this problem seems to have a very high potential.
Marine algae have the ability to accumulate halogens and produce a variety of halogenated compounds of primary importance in the biochemical cycle of halogens. Particulary, Laminaria may concentrate iodine up to 150,000 from the concentration found in seawater. Nevertheless, the basic mechanisms involved in the metabolism of halogens in algae and the biological signifiance of the halide pool in the algae are still unknown. We have chosen the brown algae Laminaria digitata (L.) Lamouroux as a biological model for characterizing, at both biochemical and molecular level., the key enzymes regulating halogen metabolism : the haloperoxydases, possessing a vanadate ion as cofactor (vHPO).
In L. digitata, these enzymes form two distinct multigfenic families : the bromoperoxydase (vBPOs) and the iodoperoxydase (IPOs). Protein extracts of both vIPO and vBPO were purified to electrophoretic homogeneity. These enzymes differ in their respective molecular weight, immulogical characteristics, peptide profiles and above all by their specific activity. While vBPO can oxidise both bromide and iodide, vIPO is specific to iodide and its oxidative capacity for this halide is higher than vBPO. This particularity suggests that vIPO, localised in the cell wall and at the level of the cortex, may be directly responsible for the absorption of iodide in this algae.
This strict specificity of vIPO compared to other vHPOs is suggested to result from an increase in electronegative potential at the active site, together with an alteration of the structural topology of the site of fixation of halides. From an evolutionary perspective, one vBPO may have been the ancestor of contemporary vHPOs in brown algae. In that case, the loss of the bromide-oxidising function may have been at the origin of vIPOs and the appearance of this novel biochemical function.
As well as the particular and specific role of vIPOs in the absorption of iodine, members of both vHPO families seem to be involved in defense mechanisms and some stress induced responses and particularly under oxidative stress conditions. Within this context, they play a direct role as anti-oxidative enzymes, by consuming hydrogen peroxide. They may also synthesise halogenated compounds which are considered as defence mechanisms. We have also established the existence of some links between their induction with the oxylipin pathway.
This chapter focusses on the properties of the novel vanadium-containing bromoperoxidases. Vanadium has been shown to be an essential requirement in the biological systems and the chemistry of the element is gaining considerable interest. As the chemistry of vanadium is of direct relevance to the mechanism of action of bromoperoxidases, the chemistry of vanadate, properties of vanadium (V) complexes, and their reactivity, including reactions with peroxide, are discussed in the chapter. The work in three selected areas, vanadium in mushrooms, vanadium in oil and coal, and vanadium in tunicates, are also discussed in the chapter. Vanadium is also an essential element for some marine macro-algae, such as the brown seaweed F. spiralus and the green seaweed Enteromorpha compressa. Most assay methods to detect bromoperoxidase activity are based on the bromination of monochlorodimedone, a cyclic diketone that has high affinity for HOBr. The steady-state kinetics of the reaction of vanadium bromoperoxidase with hydrogen peroxide and bromide has been extensively studied. Hypobromous acid is known to be in rapid equilibrium with molecular bromine and tribromide ions in aqueous solutions. The first electron paramagnetic resonance (EPR) spectrum reported of an extract of the cap of the mushroom showed clearly an EPR signal characteristic of oxo-vanadium (IV). Tunicates, commonly called “sea squirts,” are very successful marine organisms found in all oceans of the world.
IntroductionEnzymatic Catalytic PromiscuityDesign of New Enzyme Catalyzed ReactionsSummary and OutlookReferences
Undecaprenyl Phosphate Synthesis, Page 1 of 2
Abstract
Undecaprenyl phosphate (C55-P) is an essential 55-carbon long-chain isoprene lipidinvolved in the biogenesis of bacterial cell wall carbohydrate polymers: peptidoglycan, O antigen, teichoic acids, and other cell surface polymers. It functions as a lipid carrier that allows the traffic of sugar intermediates across the plasma membrane, towards the periplasm,where the polymerization of the different cellwall components occurs. At the end of these processes, the lipid is released in a pyrophosphate form (C55-PP). C55-P arises from the dephosphorylation of C55-PP, which itself originates from either a recycling event or a de novo synthesis. In Escherichia coli, the formation of C55-PP is catalyzed by the essential UppS synthase, a soluble cis-prenyltransferase, whichadds eight isoprene units ontofarnesyl pyrophosphate. Severalapo- and halo-UppSthree-dimensional structures have provided a high level of understanding of this enzymatic step. The following dephosphorylationstep is required before the lipid carrier can accept a sugar unit at the cytoplasmic face of the membrane. Four integralmembrane proteins have been shown to catalyzethis reaction in E. coli:BacA and three members of the PAP2 super-family:YbjG, LpxT, and PgpB. None of these enzymes is essential,but the simultaneous inactivation of bacA, ybjG, and pgpB genes gave rise to a lethal phenotype, raising the question of the relevance of such a redundancy of activity. It was alsorecently shown that LpxTcatalyzes the specific transfer of the phosphate group arising from C55-PP to the lipidA moiety of lipopolysaccharides, leading to a lipid-A 1-diphosphate form whichaccounts for one-third of the total lipidA in wild-type E. coli cells. The active sites of LpxT, PgpB,andYbjG were shown to face the periplasm, suggesting that PAP2 enzymes arerather involved in C55-PP recycling. These recent discoveries have opened the way to the elucidation of the functional and structural characterization of these different phosphatases.
Copper complexes [Cu2(μ2-C2O4)(HC2O4)(L1)]·(H2C2O4) (1), [Cu2(Bpz∗T-OEt)2Cl2CuCl4] (Bpz∗T-EtOH = L2) (2) and [Cu2(Bpz∗T-O)4]·4pz∗·4H2O (3) (Bpz∗T-O = L3) (L1 = 2,4-bis(3,5-dimethyl-1H-pyrazol-1-yl)-6-diethylamino-1,3,5-triazine, Bpz∗T = 2,4,6-tri(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine) have been synthesized in the reaction of Cu(Ac)2·2H2O, oxalic acid, L1 (for 1) or Bpz∗T(for 2 and 3) and CuCl2·2H2O with solution methods. They were characterized by elemental analysis, IR, UV-vis and thermogravimetric analyses (TG), and the single-crystal X-ray diffraction analysis. Structural analysis reveals that centre metal Cu atoms in the complexes 1 and 3 are six-coordination modes, forming distorted octahedron geometries with N2, O2 and N6 donors, respectively. While Cu atom in the complex 2 is four-coordination and five-coordination modes, forming tetragonal pyramid and tetrahedron geometry with N3 Cl2 and N6, respectively. The complexes exhibit catalytic bromination activity in a single-pot reaction of the conversion of phenol red to bromophenol blue in a mixed system of H2O-DMF-KBr at the constant temperature of 30 ± 0.5 C with a buffer solution of NaH2PO4-Na2HPO4 (pH=5.8), indicating that they can be considered as a potential functional model of bromoperoxidase.
Saccharina is one of the most important cold-water living marine brown algal genera. In this study we analyzed the transcriptome of S. japonica, which belongs to the 1 000 Plants (OneKP) Project, by using a next-generation high-throughput DNA sequencing technique. About 5.16 GB of raw data were generated, and 65 536 scaffolds with an average length of 454 bp were assembled with SOAP de novo assembly method. In total, 19 040 unigenes were identified by BLAST; 25 734 scaffolds were clustered into 37 Gene ontology functional groups; 6 760 scaffolds were classified into 25 COG categories, as well as 2 665 scaffolds that were assigned to 306 KEGG pathways. Majority of the unigenes exhibited more similarities to algae including brown algae and diatom than other cyanobacteria, marine diatom, and plant. Saccharina japonica has the outstanding capability to accumulate halogen such as Br and I via halogenation processes from seawater. We acquired 42 different vanadium-dependent haloperoxidases (vHPO) in S. japonica transcriptome data, including 5 segments of vanadium-dependent iodoperoxidase (vIPO) and 37 segments of vanadium-dependent bromoperoxidase (vBPO). Complicated analyses of identified fulllength S. japonica vBPO1 and S. japonica vBPO2 revealed the importance of vBPO among species of brown algae and the strong relationship between marine algal vBPOs and vIPOs. This study will enhance our understanding of the biological characteristics and economic values of S. japonica species.
A catalytic chlorination of ketones was performed by using a vanadium catalyst in the presence of Bu4NI and AlCl3 under atmospheric molecular oxygen. This catalytic chlorination could be applied to the chlorination of alkenes to give the corresponding vic-dichlorides. AlCl3 was found to serve as both a Lewis acid and a chloride source to induce the facile chlorination. A combination of Bu4NI and AlI3 in the presence of a vanadium catalyst under atmospheric molecular oxygen induced the iodination of ketones.
Copyright © 2015 Elsevier Inc. All rights reserved.
Vanadium-dependent haloperoxidases belong to a class of vanadium enzymes that may have potential industrial and pharmaceutical applications due to their high stability. In this study, the 5'-flanking genomic sequence and complete reading frame encoding vanadium-dependent bromoperoxidase (GcVBPO1) was cloned from the red seaweed, Gracilaria changii; and the recombinant protein was biochemically characterized. The deduced amino acid sequence of GcVBPO1 is 1818 nucleotides in length, sharing 49% identity with the vanadium-dependent bromoperoxidases from Corralina officinalis and Cor. pilulifera, respectively. The amino acid residues associated with the binding site of vanadate cofactor were found to be conserved. The Km value of recombinant GcVBPO1 for Br(-) was 4.69mM, while its Vmax was 10.61μkatmg(-1) at pH 7. Substitution of Arg(379) with His(379) in the recombinant protein caused a lower affinity for Br(-), while substitution of Arg(379) with Phe(379) not only increased its affinity for Br(-) but also enabled the mutant enzyme to oxidize Cl(-). The mutant Arg(379)Phe was also found to have a lower affinity for I(-), as compared to the wild-type GcVBPO1 and mutant Arg(379)His. In addition, the Arg(379)Phe mutant has a slightly higher affinity for H2O2 compared to the wild-type GcVBPO1. Multiple cis-acting regulatory elements associated with light response, hormone signaling, and meristem expression were detected at the 5'-flanking genomic sequence of GcVBPO1. The transcript abundance of GcVBPO1 was relatively higher in seaweed samples treated with 50 parts per thousand (ppt) artificial seawater (ASW) compared to those treated in 10 and 30ppt ASW, in support of its role in the abiotic stress response of seaweed.
In an attempt to mimic the active sites of the anion-binding enzymes vanadium haloperoxidase and acid phosphatase, two tripodal receptors have been shown to bind phosphate and vanadate anions in organic solvents through H-bonding interactions.
Mit der Entdeckung hochstereoselektiver Enzyme mit breiter Substratspezifität hat sich die Biokatalyse in den letzten Jahrzehnten rasant entwickelt. Ein neues Forschungsgebiet beschäftigt sich mit Enzymen, die eine breite Reaktionsspezifität bei der Katalyse alternativer Reaktionen aufweisen. Oft unterschätzt, spielt diese katalytische Promiskuität (catalytic promiscuity) eine natürliche Rolle in der Evolution und in manchen Fällen auch in der Biosynthese von Sekundärmetaboliten. In diesem Kurzaufsatz werden Beispiele für katalytische Promiskuität mit aktuellen und möglichen Anwendungen in der Synthese vorgestellt. Kombiniert mit Protein-Engineering könnten dank dieser Eigenschaft die Verwendungsmöglichkeiten von Enzymen in der organischen Synthese deutlich erweitert werden.
Introduction and Thermodynamic Considerations Biological Oxygen Atom Transfer Chemical Oxygen Atom Transfer Conclusion References
Protein kinase B/Akt plays a critical role in the regulation of cardiac hypertrophy, angiogenesis and apoptosis. The evidences
that elevation of Akt in cardiomyocytes in vivo and in vitro protects against apoptosis after ischemia/reperfusion injury provide possibility that agents targeting Akt activation become
a novel therapeutic strategy for limiting myocardial injury following ischemia. Vanadium compounds inhibiting protein tyrosine
phosphatases are potent activator of the Akt signaling pathways and elicit cardioprotection in heart ischemia/reperfusion
injury along with cardiac functional recovery in rats. In addition, vanadium compounds has strong anti-hypertrophic in the
pressure overload-induced hypertrophy in ovariectomized and aortic-banded rats. The elevation of Akt activity and Akt-dependent
eNOS phosphorylation are central roles on vanadium compound-induced anti-hypertrophy and heart failure in the ovariectomized
and aortic-banded rats. Taken together, vanadium compounds are potential therapeutics for ischemia/reperfusion-induced myocardial
injury and heart failure associated with hypertension in the postmenopausal women.
KeywordsCardiovascular disease-Hypertrophy-Endothelial nitric oxide synthase-Postmenopausal women-Protein kinase B-Protein tyrosine phosphatase-Vanadium compounds
Vanadium haloperoxidases contain the bare metal oxide vanadate as a prosthetic group and differ strongly from the heme peroxidases
in substrate specificity and molecular properties. The substrates of these enzymes are limited to halides and sulfides, which
in the presence of hydrogen peroxide are converted into hypohalous acids or sulfoxides, respectively. Several seaweeds contain
iodo- and bromoperoxidases and their direct or indirect involvement in the production of the huge amounts of brominated, iodinated
compounds and the formation of I2 in the marine environment will be reviewed. Vanadium chloroperoxidases occur in a group of common terrestrial fungi and are
probably involved in the degradation of plant cell walls and breakdown of the leaf cuticle. The natural presence of high-molecular-weight
chloro-aromatics in the environment is probably be due to the activity of these enzymes. Based upon several X-ray structures
of the enzymes and detailed kinetics a molecular mechanism is proposed and discussed in detail. As will be shown the metal
oxide in the active site binds hydrogen peroxide in a side-on fashion and acts as a Lewis acid allowing nucleophilic attack
of an incoming halide and formation of HOX. The surprising evolutionary relationship between the bacterial and mammalian acid
phosphatases that hydrolyze phosphate monoesters and the vanadium haloperoxidases will be shown.
KeywordsVanadium iodoperoxidases-Vanadium bromoperoxidases-Vanadium chloroperoxidase-Steady-state kinetics-X-ray structures-Active site structures-Mutants-Sulfoxidation-Acid phosphatases-Halometobolites-Biological importance-Environmental significance-Atmospheric chemistry
The oxidative bromination of arenes is induced by a vanadium catalyst in the presence of a bromide salt and a Brønsted acid
or a Lewis acid under molecular oxygen, which provides an eco-friendly bromination method as compared with a conventional
bromination one with bromine. This catalytic bromination can be applied to the bromination of alkenes and alkynes to give
the corresponding vic-dibromides. Use of aluminium halide as a Lewis acid in place of a Brønsted acid is demonstrated to provide a more practical
protocol for the oxidative bromination. From ketones, α-bromination products are obtained. AlBr3 is found to serve as both a bromide source and a Lewis acid for the smooth bromination reaction.
KeywordsVanadium bromoperoxidase-Vanadium catalyst-Oxidative bromination-Molecular oxygen-Brønsted acid-Lewis acid
Marine red algae are important organisms from both an ecological and an economical point of view. In most subtidal or intertidal
habitats, their extremely high diversity contributes to the functioning of the ecosystems, and in coral reef ecosystems, coralline
red algae play a major role in reef building. Red algae have also provided the resources to establish a fruitful aquaculture
in Far East Asia, first in Japan with the development of nori cultivation since the eighteenth century and most recently in
the Philippines, Indonesia and East Africa, with the farming of carrageenophytes Eucheuma and Kappaphycus) promoted by Maxwell
Doty during the 1970s (Ask and Azanza, 2002).
Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent haloperoxidases.
The steady state kinetic mechanism of the bromide-assisted disproportionation of hydrogen peroxide, forming dioxygen, catalyzed by vanadium bromoperoxidase has been investigated and compared to the mechanism of monochlorodimedone (MCD) bromination under conditions of 0.0125-6 mM H2O2, 1-500 mM Br-, and pH 4.55-6.52. Under these conditions, 50 microM MCD was sufficient to inhibit at least 90% of the dioxygen formation during MCD bromination. The rate data is consistent with a substrate-inhibited Bi Bi Ping Pong mechanism, in which the substrate bromide, is also an inhibitor at pH 4.55 and 5.25, but not at pH 5.91 and 6.52. The kinetic parameter KmBr, KmH2O2, KisBr, and KiiBr determined for the reactions of bromide-assisted disproportionation fo hydrogen peroxide and MCD bromination are similar, indicating that the mechanisms of both reactions occur via the formation of a common intermediate, the formation of which is rate-limiting. Fluoride is a competitive inhibitor with respect to hydrogen peroxide in both reactions at pH 6.5. At high concentrations of hydrogen peroxide, the bromide-assisted disproportionation of hydrogen peroxide occurs during the bromination of MCD. The sum of the rates of MCD bromination and dioxygen formation during MCD bromination is equal to the rate of dioxygen formation in the absence of MCD. The apportionment of the reaction through the MCD bromination and dioxygen formation pathways depends on pH, with much lower hydrogen peroxide concentrations causing significant dioxygen formation at higher pH.
The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed
mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding
residue His496 is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme.
The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg360, Arg490, and Lys353. When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can
still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the
kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic, and statistical refinements permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is described for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position Specific Iterated BLAST (PSLBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities.
The reaction of vanadium-bromoperoxidase from the brown alga Ascophyllum nodosum with hydrogen peroxide, bromide, and 2-chlorodimedone has been subjected to an extensive steady-state kinetic analysis. Systematic variation of pH and the concentrations of these three components demonstrate that the reaction model includes four enzyme species: native bromoperoxidase, a bromoperoxidase-bromide inhibitory complex, a bromoperoxidase-hydrogen peroxide intermediate, and a bromoperoxidase-HOBr species. This latter intermediate did not display any direct interaction with the nucleophilic reagent as oxidized bromine species (Br-3, Br2, and/or HOBr) were the primary reaction products. The generation of oxidized bromine species was as fast as the bromination of 2-chlorodimedone. The enzyme did not show any specificity with regard to bromination of various organic compounds. Formation of the bromoperoxidase-bromide inhibitory complex was competitive with the reaction between hydrogen peroxide and enzyme. From the steady-state kinetic data lower limits for the second-order rate constants at various pH values were calculated for individual steps in the catalytic cycle. This pH study showed that native enzyme must be unprotonated prior to binding of hydrogen peroxide (second-order association rate constant of 2.5.10(6) M-1.s-1 at pH greater than 6). The pKa for the functional group controlling the binding of hydrogen peroxide was 5.7 and is ascribed to a histidine residue. The reaction rate between bromide and enzyme-hydrogen peroxide intermediate also depended on pH (second-order association rate constant of 1.7.10(5) M-1.s-1 at pH 4.0).
Bromoperoxidase was purified from the crude extract of Corallina pilulifera to be homogeneous upon polyacrylamide disc gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses according to the procedures previously reported (Itoh, N., Izumi, Y., and Yamada, H. (1985) Biochem. Biophys. Res. Commun. 131, 428-435). The enzyme had a molecular weight of approximately 790,000 and was composed of 12 subunits of identical molecular weights (Mr 64,000). Hexagonal molecular shapes of the enzyme were observed by electron microscopy. The isoelectric point of the enzyme was 3.0, and the predominance of acidic amino acids was revealed by amino acid analysis of the enzyme. The enzyme was specific for I- and Br- and inactive toward Cl- and F-. The optimum pH of the enzyme was 6.0, and the enzyme was stable in a range from pH 5.0 to 11.0. The enzyme had no hemeor flavin-like compounds as a prosthetic group. Plasma emission spectroscopy revealed that the enzyme contains 2.3 +/- 0.2 atoms of iron and 1.6 +/- 0.1 atoms of magnesium/molecule of protein. Hence, bromoperoxidase of C. pilulifera was distinct from other haloperoxidases and many peroxidases, which are hemoproteins.
Using reverse transcription of messenger RNA followed by amplification using the polymerase chain reaction, three overlapping cDNA fragments encompassing the encoding sequence of the vanadium chloroperoxidase from the fungus Curvularia inaequalis were isolated and sequenced. The sequence was confirmed by DNA sequence analysis of genomic DNA. The deduced amino acid sequence predicts a protein of 609 residues with a mass of 67488 Da. Competitive reverse-transcription polymerase chain reaction analysis indicates that vanadium chloroperoxidase expression takes place in the secondary-growth phase initiated by nutrient depletion. Southern-blot analysis of genomic DNA indicates that there is only a single gene encoding the vanadium chloroperoxidase and that no isoenzymes are present. The N-terminal amino acid residue was blocked and could not be determined by amino acid sequencing, although evidence is presented showing that the N-terminal region starts very close to the first encoded methionine residue. Although the vanadium chloroperoxidase is secreted, it was not possible to assign a leader peptide. The protein contains two putative N-glycosylation sites but experiments indicate that the protein is non-glycosylated. Two cysteine residues are present in the protein both as free thiols: no disulphide bridging was found. Metal analysis revealed that iron, copper, and calcium do not constitute part of the protein. Zinc was found at a ratio of 0.3 +/- 0.04 mol/mol protein. Boiling and subsequent SDS/PAGE of the protein sample showed a typical degradation pattern of the enzyme. Amino acid sequence analysis of the resulting peptides showed that the cleavage took place at Asp-Pro bonds of which six are located throughout the protein. No sequence similarity with other known peroxidases was found except for one small region, sharing limited similarity with bacterial haloperoxidases and other alpha/beta-hydrolase-fold enzymes. In the case of the bacterial bromoperoxidases from this group, a methionine located in this region was suggested to have a role in catalysis. Methionine, however, was not involved in the catalysis of the vanadium chloroperoxidase.
The chloroperoxidase (EC 1.11.1.-) from the fungus Curvularia inaequalis belongs to a class of vanadium enzymes that oxidize halides in the presence of hydrogen peroxide to the corresponding hypohalous acids. The 2.1 A crystal structure (R = 20%) of an azide chloroperoxidase complex reveals the geometry of the catalytic vanadium center. Azide coordinates directly to the metal center, resulting in a structure with azide, three nonprotein oxygens, and a histidine as ligands. In the native state vanadium will be bound as hydrogen vanadate(V) in a trigonal bipyramidal coordination with the metal coordinated to three oxygens in the equatorial plane, to the OH group at one apical position, and to the epsilon 2 nitrogen of a histidine at the other apical position. The protein fold is mainly alpha-helical with two four-helix bundles as main structural motifs and an overall structure different from other structures. The helices pack together to a compact molecule, which explains the high stability of the protein. An amino acid sequence comparison with vanadium-containing bromoperoxidase from the seaweed Ascophyllum nodosum shows high similarities in the regions of the metal binding site, with all hydrogen vanadate(V) interacting residues conserved except for lysine-353, which is an asparagine.
We show here that the amino acid residues contributing to the active sites of the vanadate containing haloperoxidases are conserved within three families of acid phosphatases; this suggests that the active sites of these enzymes are very similar. This is confirmed by activity measurements showing that apochloroperoxidase exhibits phosphatase activity. These observations not only reveal interesting evolutionary relationships between these groups of enzymes but may also have important implications for the research on acid phosphatases, especially glucose-6-phosphatase-the enzyme affected in von Gierke disease-of which the predicted membrane topology may have to be reconsidered.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
The crystal structure of Dps, a DNA-binding protein from starved E. coli that protects DNA from oxidative damage, has been solved at 1.6 A resolution. The Dps monomer has essentially the same fold as ferritin, which forms a 24-mer with 432 symmetry, a hollow core and pores at the three-fold axes. Dps forms a dodecamer with 23 (tetrahedral) point group symmetry which also has a hollow core and pores at the three-folds. The structure suggests a novel DNA-binding motif and a mechanism for DNA protection based on the sequestration of Fe ions.
A new crystal form of the vanadium-dependent bromoperoxidase from Corallina officinalis has been obtained. The crystals exhibit a 'teardrop' morphology and are grown from 2 M ammonium dihydrogen phosphate pH and diffract to beyond 1.7 A resolution. They are in tetragonal space group P4222 with unit-cell dimensions of a = b = 201.9, c = 178.19 A, alpha = beta = gamma = 90 degrees. A 2.3 A resolution native data set has been collected at the Hamburg Synchrotron. A mercury derivative data set has also been collected, and the heavy-atom positions have been determined. The self-rotation function and the positions of the heavy atoms are consistent with the molecule being a dodecamer with local 23 symmetry.
The structure of Escherichia blattae non-specific acid phosphatase (EB-NSAP) has been determined at 1.9 A resolution with a bound sulfate marking the phosphate-binding site. The enzyme is a 150 kDa homohexamer. EB-NSAP shares a conserved sequence motif not only with several lipid phosphatases and the mammalian glucose-6-phosphatases, but also with the vanadium-containing chloroperoxidase (CPO) of Curvularia inaequalis. Comparison of the crystal structures of EB-NSAP and CPO reveals striking similarity in the active site structures. In addition, the topology of the EB-NSAP core shows considerable similarity to the fold of the active site containing part of the monomeric 67 kDa CPO, despite the lack of further sequence identity. These two enzymes are apparently related by divergent evolution. We have also determined the crystal structure of EB-NSAP complexed with the transition-state analog molybdate. Structural comparison of the native enzyme and the enzyme-molybdate complex reveals that the side-chain of His150, a putative catalytic residue, moves toward the molybdate so that it forms a hydrogen bond with the metal oxyanion when the molybdenum forms a covalent bond with NE2 of His189.
The equilibria in the system l-histidine+VO2+ in aqueous solution have been studied in the pH range 2–13 by a combination of pH potentiometry and spectroscopy (ESR, visible absorption and circular dichroism). The results of the various methods are made self-consistent, then rationalized assuming an equilibrium model including species (where HL denotes l-histidine): MLH2, MLH, MLH−2, ML2H4, ML2H3, ML2H2, ML2H, ML2, ML2H−1, M2L2H−4 and several products of hydrolysis; formation constants, absorption and circular dichroism spectra are given for each species. Plausible isomeric structures for each stoichiometry in solution are discussed.
At physiological pH and ionic strength, histidine (H-His-OH) and vanadate form at least two weak complexes with the following characteristics: δ(51V) (ppm) (K (M−1)): 1 −546 (0.7), 2 −561 (1.6). K is the formation constant, and a 1:1 stoichiometry with the ligand coordinating through the amino group is assumed. Type 1 complexes are also formed with H-His-OR (R Me, Bz), but not with BOC-His-OH. An additional, very weak complex 3 (δ = −591 ppm at pH 7.2, −573 ppm at pH 8.8) is present at c(V)>5 mM. α-Alanyl-histidine gives rise to a relatively strong, possibly dinuclear monoligate complex 4 (δ(51V) = −519 ppm, K = 5×105 M−2 at pH 7.2). Crucial for the formation of 4 are the deprotonated amide of the peptide linkage, the NH2 group (as part of a chelate-5 ring; β-alanyl-histidine does not provide a type 4 complex), and the carboxylate unit. The complex stoichiometries and modes of coordination have been evaluated on the basis of 13C, 14N and quantitative 51V NMR spectroscopy.
An electron paramagnetic resonance (EPR) study was carried out to examine structural aspects of vanadium-containing bromoperoxidase from the brown seaweed Ascophyllum nodosum. At high pH, the reduced form of bromoperoxidase showed an apparently axially symmetric EPR signal with 16 hyperfine lines. When the pH was lowered, a new EPR spectrum was formed. When EPR spectra of the reduced enzyme were recorded in the pH range from 4.2 to 8.4, it appeared that these changes were linked to a functional group with an apparent pK/sub a/ of about 5.4. In DâO this value for the pK/sub a/ was 5.3. It is suggested that these effects arise from protonation of histidine or aspartate/glutamate residues near the metal ion. The values for the isotropic hyperfine coupling constant of the reduced enzyme at both high and low pH are also consistent with a ligand field containing nitrogen and/or oxygen donor atoms. When reduced bromoperoxidase was dissolved in DâO or Hâ¹â·O instead of Hâ¹â¶O, vanadium (IV) hyperfine line widths were markedly affected, demonstrating that water is a ligand of the metal ion. Together with previous work these findings suggest that vanadium (IV) is not involved in catalytic turnover and confirm the model in which the vanadium (V) ion of the native enzyme only serves to bind both hydrogen peroxide and bromide. After excess vanadate was added to a homogeneous preparation of purified bromoperoxidase, the extent of vanadium bound to the protein increased from 0.5 to 1.1, with a concomitant enhancement of enzymic activity. Finally, it is demonstrated that both vanadate (VOâ/sup 3 -/) and molybdate (MoOâ/sup 2 -/) compete for the same site on apobromoperoxidase.
The 17O NMR of bromoperoxidase in Tris buffer at pH 8 treated with 17O-enriched H2O2 reveals direct binding of peroxide to active site vanadium both in the symmetric and asymmetric modes, the latter possibly due to hydroperoxide. In addition, non-active site HVO2(O2)22− is detected. The results are counter-checked with NMR data on peroxovanadium model compounds.
Bromine K-edge EXAFS studies have been carried out for bromide/peroxidase samples in Tris buffer at pH 8. The results are compared with those of aqueous (Tris-buffered) bromide and vanadium model compounds containing Br-V, Br-C(aliphatic) and Br-C(aromatic) bonds. It is found that bromide does not coordinate to the vanadium centre. Rather, bromine binds covalently to carbon. A possible candidate is active site serine.
The kinetics of bromination of phenol red (HPhR) to yield bromophenol blue (BrPhB) was studied at pH 6.5, in the presence of peroxovanadium(V) species generated by acid decomposition of [VO(O2)2(NH3)]− and of [O{VO(O2)2}2]4−. In the concentration ranges 10−6–10−7 (HPhR), (1.5–8.0) × 10−4 (vanadium complexes) and 0.004–0.12 mol dm−3 (bromide), the rate law is R = k[V]T [Br−][HS], where HS is the substrate undergoing bromination in the rate determining step, with k = 2.49 × 105 dm6 mol−2 s−1. Acid treatment of the precursor complexes yields a mixture of [VO(O2)L]n complexes, with L = H2O, [VO(O2)(H2O)]+, or O22−, [VO(O2)2]−. Alkalinization leads to active species that react with bromide to yield a brominated vanadium complex (e.g. [VO(O2)Br]), which is postulated to be the active bromination agent. Kinetic data rule out the mediation of hypobromous acid. The results support the idea that five-co-ordinated vanadium species are required in the bromination reaction.
In the prepurified extract from Ascophyllum nodosum (PEX-A), it was possible to detect the peroxidase-de-pendent iodination of various compounds using an iodide-sensitive electrode. Cyanide did not significantly inhibit peroxidase-dependent reactions in the algal extract in contrast to the iodination with the hemoproteide lactoperoxidase. Iodo-phloroglucinols and iodo-4-hydroxybenzylalcohols were isolated and identified in semi-preparative test samples. Chlorination of l,l-dimethyl-4-chloro-3,5-cyclohexadione was not observed, but it was possible to detect bromination of this substance. Bromo-4-hydroxybenzylalcohols could not be obtained. It was only possible to record little catalase activity. This could possibly be attributed to the secondary activity of peroxidase activity.
Bromoperoxidase from Ascophyllum nodusum was the first vanadium-containing enzyme to be isolated. X-ray absorption spectra have now been collected in order to investigate the coordination of vanadium in the native, native plus bromide, native plus hydrogen peroxide, and dithionite-reduced forms of the enzyme. The edge and X-ray absorption near-edge structures show that, in the four samples studied, it is only on reduction of the native enzyme that the metal site is substantially altered. In addition, these data are consistent with the presence of vanadium(IV) in the reduced enzyme and vanadium(V) in the other samples. Extended X-ray absorption fine structure data confirm that there are structural changes at the metal site on reduction of the native enzyme, notably a lengthening of the average inner-shell distance, and the presence of terminal oxygen together with histidine and oxygen-donating residues.
Haloperoxidases are enzymes which catalyze the oxidation of halide ions (i.e. chloride, bromide and iodide) by hydrogen peroxide. These enzymes usually contain the FeHeme moiety or vanadium as an essential constituent at their active site, however, a few haloperoxidases which lack a metal cofactor are known. This review will examine (1) the reactivity of the vanadium haloperoxidases, particularly the mechanism of halide oxidation by hydrogen peroxide, and the mechanism of halogenation and sulfoxidation, including the newly reported regioselectivity and enantioselectivity of the vanadium haloperoxidases; (2) the X-ray structure of vanadium chloroperoxidase, the vanadium(V) active site and the role of critical amino acid side chains for catalysis and (3) functional biomimetic systems, with specific relevance to the mechanism of the vanadium haloperoxidase enzymes.
A series of oxoperoxovanadium(V) complexes (ligands: H3nta = nitrilotriacetic acid, H3heida = N-(2-hydroxyethyl)iminodiacetic acid, H2ada = N-(2-amidomethyl)iminodiacetic acid, Hbpg = N,N-bis(2-pyridylmethyl)glycine, and tpa = N,N,N-tris(2-pyridylmethyl)amine) were characterized as functional models for the vanadium haloperoxidase enzymes. The crystal structures of K[VO(O2)Hheida], K[VO(O2)ada], [VO(O2)bpg], and H[VO(O2)bpg]2(ClO4) were obtained. These complexes all possess a distorted pentagonal bipyramidal coordination sphere containing a side-on bound peroxide. In the presence of sufficient acid equivalents these complexes catalyze the two-electron oxidation of bromide or iodide by peroxide. Halogenation of an organic substrate was demonstrated by following the visible conversion of Phenol Red to Bromophenol Blue. In the absence of substrate, dioxygen can be generated by the halide-assisted disproportionation of hydrogen peroxide. In addition, some of these complexes can efficiently catalyze the peroxidative halogenation reaction, performing multiple turnovers in minutes. The kinetic analysis of the halide oxidation reaction indicates a mechanism which is first order in protonated peroxovanadium complex and halide. The bimolecular rate constants for both bromide and iodide oxidation were determined, with the iodide rates being approximately 5−6 times faster than the bromide rates. The rate constants obtained for bromide oxidation range from a maximum of 280 M-1 s-1 for the Hheida complex to a minimum of 21 M-1 s-1 for the Hbpg complex. The pKa of activation for each complex in acetonitrile was determined to range from 5.4 to 6.0. On the basis of the chemistry observed for these model compounds, a mechanism of halide oxidation and a detailed catalytic cycle are proposed for the vanadium haloperoxidase enzyme.
The enzymatic halogenation of nitrogen-containing aromatic heterocycles catalyzed by two different types of haloperoxidases, the chloroperoxidase of Caldariomyces fumago (heme type) and the bromoperoxidase of Corallina pilulifera (non-heme type), has been studied. Chloroperoxidase catalyzed the chlorination of uracil and pyrazole, the bromination of cytosine, uracil, thymine, cytidine, 2′-deoxyuridine, guanosine, and pyrazole, and the iodination of uracil and pyrazole to yield the respective halogenated products. The bromoperoxidase also catalyzed the bromination of cytosine, uracil, cytidine, and pyrazole and the iodination of uracil and pyrazole to form the same products as in the chloroperoxidase reactions. A slight difference in the reactivity toward these substrates was observed between the two haloperoxidases. The results of product and halogenation intermediate analyses suggested that the bromination reaction of the bromoperoxidase occurs at the active site of the enzyme. On the contrary, the halogenation by the chloroperoxidase was found to involve the formation of a molecular halogen and its release into the solution. On the basis of the results, we discussed the abilities of the haloperoxidases as halogenating reagents.
Two aqua-oxovanadium complexes, viz. [A-VO(H2O)(sal-l-Leu)] (1) and [VO(H2O)2(5-Br-sal-Gly)]·H2O (2·H2O), containing the water ligands in cis- and trans-positions to the oxo group at V–OH2 distances ranging from 2.008 to 2.228 Å, have been structurally characterized in order to model the apical electron density feature found in the structures of fungal and algal vanadate-dependent peroxidases. Br K-edge XAS of bromide-treated bromoperoxidase from Ascophyllum nodosum and model compounds (including 2·H2O) has been used to show that the substrate bromide does not bind to active site vanadium but to a light atom, possibly carbon, in its vicinity.
Minireviews do not have abstracts.
The mechanism of a membrane-bound enzyme important in phospholipid signaling, type 2 phosphatidic acid phosphatase, is suggested by sequence motifs shared with a soluble vanadium-dependent chloroperoxidase of known structure. These regions are also conserved in other soluble globular and membrane-associated proteins, including bacterial acid phosphatases, mammalian glucose-6-phosphatases, and the Drosophila developmental protein Wunen. This implies that a similar arrangement of catalytic residues specifies the active site within both soluble and membrane spanning domains.
Using reverse transcription of messenger RNA followed by amplification using the polymerase chain reaction, three overlapping cDNA fragments encompassing the encoding sequence of the vanadium chloroperoxidase from the fungus Curvularia inaequalis were isolated and sequenced. The sequence was confirmed by DNA sequence analysis of genomic DNA. The deduced amino acid sequence predicts a protein of 609 residues with a mass of 67 488 Da. Competitive reverse-transcription polymerase chain reaction analysis indicates that vanadium chloroperoxidase expression takes place in the secondary-growth phase initiated by nutrient depletion. Southern-blot analysis of genomic DNA indicates that there is only a single gene encoding the vanadium chloroperoxidase and that no isoenzymes are present. The N-terminal amino acid residue was blocked and could not be determined by amino acid sequencing, although evidence is presented showing that the N-terminal region starts very close to the first encoded methionine residue. Although the vanadium chloroperoxidase is secreted, it was not possible to assign a leader peptide. The protein contains two putative N-glycosylation sites but experiments indicate that the protein is non-glycosylated. Two cysteine residues are present in the protein both as free thiols; no disulphide bridging was found. Metal analysis revealed that iron, copper, and calcium do not constitute part of the protein. Zinc was found at a ratio of 0.3 ± 0.04 mol/mol protein. Boiling and subsequent SDS/PAGE of the protein sample showed a typical degradation pattern of the enzyme. Amino acid sequence analysis of the resulting peptides showed that the cleavage took place at Asp-Pro bonds of which six are located throughout the protein. No sequence similarity with other known peroxidases was found except for one small region, sharing limited similarity with bacterial haloperoxidases and other α/β-hydrolase-fold enzymes. In the case of the bacterial bromoperoxidases from this group, a methionine located in this region was suggested to have a role in catalysis. Methionine, however, was not involved in the catalysis of the vanadium chloroperoxidase.
The purification and characterization of bromoperoxidase from the marine brown alga Laminaria saccharina is described. The purified enzyme is homogeneous, as verified by polyacrylamide gel electrophoresis and size-exclusion high-performance liquid chromatography; it has a dimeric structure with a relative molecular mass of 108 kDa. Reconstitution experiments showed that the transition metal vanadium is essential for the brominating activity of this enzyme. Analysis of bromoperoxidase with electron paramagnetic resonance in combination with activity measurements indicated that the ratio of vanadium ligated to the protein at the active site increased from 0.3 to 2.0 when the purified enzyme was incubated with an excess of the metal. Properties of bromoperoxidase from L. saccharina are compared with bromoperoxidase from Ascophyllum nodosum (De Boer, E., Van Kooyk, Y., Tromp, M.G.M., Plat, H. and Wever, R. (1986) Biochim. Biophys. Acta 869, 48–53). The presence of ‘vanadium(V)-dependent’ haloperoxidase activity in crude extracts of Fucus spiralis, F. serratus, F. vesiculosus, Pelvetia canaliculata and Chorda filum is described. These experiments demonstrate that vanadium-containing haloperoxidases are more widely spread in nature
The Pyrococcus furiosus (PF) ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100 degrees C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 A and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis-Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.
Vanadium bromoperoxidase (V-BrPO) catalyzes the oxidation of bromide by hydrogen peroxide, which results in the bromation of appropriate organic substrates or the formation of dioxygen, in the absence of an organic substrate and under certain other conditions. The mechanism of the bromide-assisted disproportionation of hydrogen peroxide catalyzed by V-BrPO, which is the reaction that forms dioxygen, has been investigated for V-BrPO isolated from two new marine algal sources, Macrocystis pyrifera and Fucus distichus. The steady state kinetic studies have been performed under conditions of 0.02-40 mM H2O2, 1-500 mM Br- and pH 4.0-8.0. The rate data is consistent with a substrate-inhibited bi bi ping pong mechanism, in which the substrate bromide, is also an inhibitor by a noncompetitive-type mechanism. Bromide inhibits V-BrPO from M. pyrifera most strongly at pH 5.0-5.5 and V-BrPO from F. distichus most strongly at pH 5.5-6.0. The steady state mechanism of the Macrocystis and the Fucus enzymes are compared to the mechanism of the bromide-assisted disproportionation of hydrogen peroxide catalyzed by V-BrPO from Ascophyllum nodosum. In addition, the substrate hydrogen peroxide can also inhibit V-BrPO.
Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
The steady state kinetic mechanism of the bromide-assisted disproportionation of hydrogen peroxide, forming dioxygen, catalyzed by vanadium bromoperoxidase has been investigated and compared to the mechanism of monochlorodimedone (MCD) bromination under conditions of 0.0125-6 mM H2O2, 1-500 mM Br-, and pH 4.55-6.52. Under these conditions, 50 microM MCD was sufficient to inhibit at least 90% of the dioxygen formation during MCD bromination. The rate data is consistent with a substrate-inhibited Bi Bi Ping Pong mechanism, in which the substrate bromide, is also an inhibitor at pH 4.55 and 5.25, but not at pH 5.91 and 6.52. The kinetic parameter KmBr, KmH2O2, KisBr, and KiiBr determined for the reactions of bromide-assisted disproportionation fo hydrogen peroxide and MCD bromination are similar, indicating that the mechanisms of both reactions occur via the formation of a common intermediate, the formation of which is rate-limiting. Fluoride is a competitive inhibitor with respect to hydrogen peroxide in both reactions at pH 6.5. At high concentrations of hydrogen peroxide, the bromide-assisted disproportionation of hydrogen peroxide occurs during the bromination of MCD. The sum of the rates of MCD bromination and dioxygen formation during MCD bromination is equal to the rate of dioxygen formation in the absence of MCD. The apportionment of the reaction through the MCD bromination and dioxygen formation pathways depends on pH, with much lower hydrogen peroxide concentrations causing significant dioxygen formation at higher pH.
With synchrotron radiation from the Bonn 2.5 GeV synchrotron, high-resolution absorption spectra have been measured at the vanadium K-edge of bromoperoxidase from the marine brown alga Ascophyllum nodosum and several model compounds. The near-edge structure (XANES) of these spectra was used to determine the charge state and the coordination geometry around the vanadium atom. For the active enzyme a coordination charge of 2.7 was found which is compatible with a formal valence of +5, assuming coordination by atoms with a high electronegativity such as oxygen or nitrogen. For the reduced enzyme the coordination charge value of 2.15 indicates the reduction of the valency by 1 unit. Our results suggest that the coordination sphere of the vanadium atom in the native enzyme consists of at least seven oxygen atoms in a distorted octahedral environment with an average bond length of about 2 A. Through the reduction process, the coordination sphere of the vanadium atom changes with a simultaneous decrease of the coordination cage. These results agree with those deduced from previous EPR and 51V-NMR measurements.
Hydrogen peroxide, which is a substrate of vanadium bromoperoxidase (V-BrPO), has been shown to be a noncompetitive inhibitor of V-BrPO. Hydrogen peroxide inhibition increases with increasing pH. The inhibition is reversible under the conditions of the initial steady-state kinetic experiments. Analysis of the inhibition constants (KiiH2O2, KisH2O2) versus H+ concentration indicates that an ionizable group with a pKa between 6.5 and 7 is involved in the inhibition. The origin of the oxygen atoms in the dioxygen produced by the V-BrPO-catalyzed bromide-assisted disproportionation of hydrogen peroxide has been shown through H2(18)O2 labeling experiments to originate from the same molecule of hydrogen peroxide. V-BrPO-catalyzed bromination is shown to be an electrophilic (Br+) as opposed to a radical (Br.) process. The stoichiometry of H2O2 consumed to MCD reacted or to O2 produced is reported. The concentration of hydrogen peroxide also affects the competition of dioxygen formation during MCD bromination; competitive dioxygen formation is strongly enhanced at high pH. Turnover of V-BrPO under conditions of very high hydrogen peroxide concentration leads to irreversible inactivation at pH 4 and pH 5. Much less inactivation occurs during turnover at long reaction times at higher pH (> pH 6), and the inactivation can be fully reversed by subsequent addition of vanadate.
The vanadium-dependent haloperoxidase from the seaweed Corallina officinalis has been purified to homogeneity and crystallised. The protein is reported to be a hexamer of 12 x 64,000 Da, contains no haem, and is dependent on vanadium for activity. The crystals are grown from polyethylene glycol (PEG) 6,000 and 0.4 M potassium chloride. They are stable and diffract to better than 2 A resolution. They are of a cubic space group I23 (or 12(1)3) with cell dimensions a = b = c = 310 A.
Implications for the catalytic mechanism of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis have been obtained from the crystal structures of the native and peroxide forms of the enzyme. The X-ray structures have been solved by difference Fourier techniques using the atomic model of the azide chloroperoxidase complex. The 2.03 A crystal structure (R = 19.7%) of the native enzyme reveals the geometry of the intact catalytic vanadium center. The vanadium is coordinated by four non-protein oxygen atoms and one nitrogen (NE2) atom from histidine 496 in a trigonal bipyramidal fashion. Three oxygens are in the equatorial plane and the fourth oxygen and the nitrogen are at the apexes of the bipyramid. In the 2.24 A crystal structure (R = 17.7%) of the peroxide derivate the peroxide is bound to the vanadium in an eta2-fashion after the release of the apical oxygen ligand. The vanadium is coordinated also by 4 non-protein oxygen atoms and one nitrogen (NE2) from histidine 496. The coordination geometry around the vanadium is that of a distorted tetragonal pyramid with the two peroxide oxygens, one oxygen and the nitrogen in the basal plane and one oxygen in the apical position. A mechanism for the catalytic cycle has been proposed based on these X-ray structures and kinetic data.
The cDNAs for a vanadium-dependent bromoperoxidase were cloned from a marine macro-alga, Corallina pilulifera. The open reading frame of one clone (bpo1) encoded a protein of 598 amino acids with a calculated molecular mass of 65312 Da in good agreement with that of 64 kDa determined for the native enzyme. The deduced amino acid sequence coincided well with partial sequences of peptide fragments of the enzyme. From the same cDNA library we also isolated another cDNA clone (bpo2) encoding a protein of 597 amino acids with an identity of about 90% to BPO1, suggesting a genetic diversity of the bromoperoxidase gene of C. pilulifera growing in a relatively narrow area. The carboxy-terminal 123 residues of the enzyme (BPO1) showed an identity of 45% to that of the marine macro-alga Ascophillum nodosum. The homology search of the sequences of bromoperoxidases from C. pilulifera (this study) and A. nodostum, and chloroperoxidase from the fungus Curvularia inaequalis indicated highly conserved sequences PxYxSGHA and LxxxxAxxRxxxGxHxxxD. Furthermore, it was found that the histidine residue directly bound to vanadium, other residues building up the metal center and catalytic histidine residue forming the active site of the chloroperoxidase from C. inaequalis are conserved in the primary structure of the bromoperoxidase from C. pilulifera. The cloned hpol was introduced into Escherichia coli, and the expressed PO1 was purified from the recombinant strain. The N-terminal amino acid sequence of the purified BPO1 was identical to the deduced sequence from the cDNA except the N-terminal methionine.
The past year has seen further structural characterisation of both nonmetal and vanadium haloperoxidase enzymes to add to that already known for the haem- and vanadium-containing enzymes. Exploitation of these enzymes for halogenation, sulfoxidation, epoxidation, oxidation of indoles and other biotransformations has increased as more information on their catalytic mechanism has been obtained.
MolScript is one of the most popular programs for the generation of publication-quality figures and the recent re-working of the program should ensure its continued popularity. However, some functionality of particular interest to crystallographers is not part of the standard program. A modified MolScript version 1.4 has been described previously, with more flexible colouring schemes among its new features. This report describes further enhancements to MolScript version 1.4, including facilities for drawing rods for helices and ribbons for oligonucleotides and allowing several formats of electron-density maps to be read and contoured using either lines or smoothed triangulated surfaces.
The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11 A resolution, respectively. The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site. At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein. The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding. The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate. The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A. The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate. The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame. When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame.
The homo-dimeric structure of a vanadium-dependent haloperoxidase (V-BPO) from the brown alga Ascophyllum nodosum (EC 1.1.11.X) has been solved by single isomorphous replacement anomalous scattering (SIRAS) X-ray crystallography at 2.0 A resolution (PDB accession code 1QI9), using two heavy-atom datasets of a tungstate derivative measured at two different wavelengths. The protein sequence (SwissProt entry code P81701) of V-BPO was established by combining results from protein and DNA sequencing, and electron density interpretation. The enzyme has nearly an all-helical structure, with two four-helix bundles and only three small beta-sheets. The holoenzyme contains trigonal-bipyramidal coordinated vanadium atoms at its two active centres. Structural similarity to the only other structurally characterized vanadium-dependent chloroperoxidase (V-CPO) from Curvularia inaequalis exists in the vicinity of the active site and to a lesser extent in the central four-helix bundle. Despite the low sequence and structural similarity between V-BPO and V-CPO, the vanadium binding centres are highly conserved on the N-terminal side of an alpha-helix and include the proposed catalytic histidine residue (His418(V-BPO)/His404(V-CPO)). The V-BPO structure contains, in addition, a second histidine near the active site (His411(V-BPO)), which can alter the redox potential of the catalytically active VO2-O2 species by protonation/deprotonation reactions. Specific binding sites for the organic substrates, like indoles and monochlordimedone, or for halide ions are not visible in the V-BPO structure. A reaction mechanism for the enzymatic oxidation of halides is discussed, based on the present structural, spectroscopic and biochemical knowledge of vanadium-dependent haloperoxidases, explaining the observed enzymatic differences between both enzymes.
Mutation studies were performed on active-site residues of vanadium chloroperoxidase from the fungus Curvularia inaequalis, an enzyme which exhibits both haloperoxidase and phosphatase activity and is related to glucose-6-phosphatase. The effects of mutation to alanine on haloperoxidase activity were studied for the proposed catalytic residue His-404 and for residue Asp-292, which is located close to the vanadate cofactor. The mutants were strongly impaired in their ability to oxidize chloride but still oxidized bromide, although they inactivate during turnover. The effects on the optical absorption spectrum of vanadium chloroperoxidase indicate that mutant H404A has a reduced affinity for the cofactor, whereas this affinity is unchanged in mutant D292A. The effect on the phosphatase activity of the apoenzyme was investigated for six mutants of putative catalytic residues. Effects of mutation of His-496, Arg-490, Arg-360, Lys-353, and His-404 to alanine are in line with their proposed roles in nucleophilic attack, transition-state stabilization, and leaving-group protonation. Asp-292 is excluded as the group that protonates the leaving group. A model based on the mutagenesis studies is presented and may serve as a template for glucose-6-phosphatase and other related phosphatases. Hydrolysis of a phospho-histidine intermediate is the rate-determining step in the phosphatase activity of apochloroperoxidase, as shown by burst kinetics.
The three-dimensional structure of the vanadium bromoperoxidase protein from the marine red macroalgae Corallina officinalis has been determined by single isomorphous replacement at 2.3 A resolution. The enzyme subunit is made up of 595 amino acid residues folded into a single alpha+beta domain. There are 12 bromoperoxidase subunits, arranged with 23-point group symmetry. A cavity is formed by the N terminus of each subunit in the centre of the dodecamer. The subunit fold and dimer organisation of the Cor. officinalis vanadium bromoperoxidase are similar to those of the dimeric enzyme from the brown algae Ascophyllum nodosum, with which it shares 33 % sequence identity. The different oligomeric state of the two algal enzymes seems to reflect separate mechanisms of adaptation to harsh environmental conditions and/or to chemically active substrates and products. The residues involved in the vanadate binding are conserved between the two algal bromoperoxidases and the vanadium chloroperoxidase from the fungus Curvularia inaequalis. However, most of the other residues forming the active-site cavity are different in the three enzymes, which reflects differences in the substrate specificity and stereoselectivity of the reaction. A dimer of the Cor. officinalis enzyme partially superimposes with the two-domain monomer of the fungal enzyme.
A virulence-associated ATP diphosphohydrolase activity in the periplasm of Shigella, identified as apyrase, was found to be markedly similar to bacterial non-specific acid phosphatases in primary structure. When the Shigella apyrase sequence was threaded in to the recently published 3D structure of the highly similar (73%) Escherichia blattae acid phosphatase it was found to have a highly overlapping 3D structure. Our analysis, which included assays for phosphatase, haloperoxidase and catalase activities, led us to hypothesize that Shigella apyrase might belong to a new class of pyrophosphatase originating as one more variant in the family of bacterial non-specific acid phosphatases. It revealed interesting structure-function relationships and probable roles relevant to pathogenesis.