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High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus
Abstract
Both the phosphatidylinositol-hydrolysing and the phosphatidylcholine-hydrolysing phospholipases C have been implicated in the generation of second messengers in mammalian cells. The phosphatidylcholine-hydrolysing phospholipase C (PLC) from Bacillus cereus, a monomeric protein containing 245 amino-acid residues, is similar to some of the corresponding mammalian proteins. This, together with the fact that the bacterial enzyme can mimic the action of mammalian PLC in causing, for example, enhanced prostaglandin biosynthesis, suggests that B. cereus PLC can be used as a model for the hitherto poorly characterized mammalian PLCs. We report here the three-dimensional structure of B. cereus PLC at 1.5 A resolution. The enzyme is an all-helix protein belonging to a novel structural class and contains, at least in the crystalline state, three Zn2+ in the active site. We also present preliminary results from a study at 1.9 A resolution of the complex between PLC and inorganic phosphate (Pi) which indicate that the substrate binds directly to the metal ions.
... LmPC-PLC has 40% amino acid sequence identity with the homolog from Bacillus cereus (Bc), BcPC-PLC, and 22% with the catalytic N-terminal domain of PC-PLC from Clostridium perfringens (Cp), CpPC-PLC 23 . Their active sites were proposed to harbor three Zn 2+ ions, coordinated by highly conserved Zn 2+ -binding amino acid residues [24][25][26][27] ( Supplementary Fig. 1). The crystal structures of Bc- 24,28 and CpPC-PLC 25,29 show a globular α-helical folding of the phospholipase domain, while the structure of LmPC-PLC has not yet been determined. ...
... Their active sites were proposed to harbor three Zn 2+ ions, coordinated by highly conserved Zn 2+ -binding amino acid residues [24][25][26][27] ( Supplementary Fig. 1). The crystal structures of Bc- 24,28 and CpPC-PLC 25,29 show a globular α-helical folding of the phospholipase domain, while the structure of LmPC-PLC has not yet been determined. ...
... Three metal ions were found bound to the active site in molA, and only two in molB (Fig. 2b). The positions of the metals ions were numbered according to the crystal structure of BcPC-PLC 24 . To show that the metal ions bound to active site of LmPC-PLC were Zn ions, we also collected data at a wavelength of 1.33 Å, where Zn should not contribute to the anomalous signal. ...
Listeriosis is one of the most serious foodborne diseases caused by the intracellular bacterium Listeria monocytogenes . Its two major virulence factors, broad-range phospholipase C ( Lm PC-PLC) and the pore-forming toxin listeriolysin O (LLO), enable the bacterium to spread in the host by destroying cell membranes. Here, we determine the crystal structure of Lm PC-PLC and complement it with the functional analysis of this enzyme. This reveals that Lm PC-PLC has evolved several structural features to regulate its activity, including the invariant position of the N-terminal tryptophan (W1), the structurally plastic active site, Zn ²⁺ -dependent activity, and the tendency to form oligomers with impaired enzymatic activity. We demonstrate that the enzymatic activity of Lm PC-PLC can be specifically inhibited by its propeptide added in trans. Furthermore, we show that the phospholipase activity of Lm PC-PLC facilitates the pore-forming activity of LLO and affects the morphology of LLO oligomerization on lipid membranes, revealing the multifaceted synergy of the two virulence factors.
... A body of evidence suggests PC-PLC activity as a human functional equivalent of prokaryotic PC-PLC. The prokaryotic PC-PLC enzyme has been isolated and purified from Bacillus cereus (PC-PLC Bc ), and its structure has been characterized ( Figure 2) [5]. PC-PLC Bc was used as a model of its mammalian counterpart, e.g., they share antigenic similarities [6] and PC-PLC Bc is able to emulate similar responses in enhancing prostaglandin biosynthesis in mammalian cells [7]. ...
... PC-PLC Bc was used as a model of its mammalian counterpart, e.g., they share antigenic similarities [6] and PC-PLC Bc is able to emulate similar responses in enhancing prostaglandin biosynthesis in mammalian cells [7]. The X-ray crystal structure is a monomeric zinc metalloproteinase with 245 amino acid residues [5]. The enzyme has thirteen α-helices with three Zn 2+ ions in the active site. ...
... A body of evidence suggests PC-PLC activity as a human functional equivalent of prokaryotic PC-PLC. The prokaryotic PC-PLC enzyme has been isolated and purified from Bacillus cereus (PC-PLCBc), and its structure has been characterized ( Figure 2) [5]. PC-PLCBc was used as a model of its mammalian counterpart, e.g., they share antigenic similarities [6] and PC-PLCBc is able to emulate similar responses in enhancing prostaglandin biosynthesis in mammalian cells [7]. ...
Phosphatidylcholine-specific phospholipase C (PC-PLC) is an enzyme that catalyzes the formation of the important secondary messengers phosphocholine and diacylglycerol (DAG) from phosphatidylcholine. Although PC-PLC has been linked to the progression of many pathological conditions, including cancer, atherosclerosis, inflammation and neuronal cell death, studies of PC-PLC on the protein level have been somewhat neglected with relatively scarce data. To date, the human gene expressing PC-PLC has not yet been found, and the only protein structure of PC-PLC that has been solved was from Bacillus cereus (PC-PLCBc). Nonetheless, there is evidence for PC-PLC activity as a human functional equivalent of its prokaryotic counterpart. Additionally, inhibitors of PC-PLCBc have been developed as potential therapeutic agents. The most notable classes include 2-aminohydroxamic acids, xanthates, N,N′-hydroxyureas, phospholipid analogues, 1,4-oxazepines, pyrido[3,4-b]indoles, morpholinobenzoic acids and univalent ions. However, many medicinal chemistry studies lack evidence for their cellular and in vivo effects, which hampers the progression of the inhibitors towards the clinic. This review outlines the pathological implications of PC-PLC and highlights current progress and future challenges in the development of PC-PLC inhibitors from the literature.
... Zinc-Metalloenzymes The B. cereus PC-PLC, C. perfringens alpha-toxin, Clostridium bifernentans PLC, Listeria monocytogenes PLC-B, and Clostridium novyi gamma-toxin form a group of related enzymes which contain essential zinc ions and are reversibly inactivated by EDTA or o-phenanthroline (54,64,76,143,160,169). It also seems likely that the phospholipases C produced from Clostridium absonum and Clostridium barati, which are antigenically and genetically related to the C. perfringens alpha-toxin (112,171), are also zinc-metallophospholipases C. ...
... Crystallographic (64) and chemical modification (5,6,86,87) studies of the B. cereus PC-PLC have provided an insight into the molecular architecture of this enzyme and, by extrapolation, the possible tertiary structures of homologous regions in the other zinc-metallophospholipases C. The enzyme is composed of seven helices, forming a twisted barrel structure. Three zinc ions, one of which is loosely bound, coordinate with amino acids from different helices and thus conformationally restrain the molecule. ...
... The active site of the B. cereus PC-PLC has been tentatively identified by cocrystallizing the protein with phosphate ions (64). This study demonstrated phosphate binding to all three zinc ions, displacing water molecules in the process. ...
A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.
... The structure of eukaryotic PC-PLC is unknown and only prokaryotic PC-PLC enzymes have been structurally characterised. The most studied PC-PLC is isolated from Bacillus cereus [17]. In this paper, we describe a search for hit compounds against human PC-PLC for potential anticancer drug development using the virtual high throughput screening (vHTS) methodology, an effective method for identifying novel inhibitors for biomolecular targets [18e21]. ...
... These compounds were filtered based on Lipinski's rules [25], resulting in 8373 drug-like molecules. The docking scaffold used was the wild-type PC-PLC Bc crystal structure (structure shown in Fig. 2) [17]. It can be seen that the catalytic site has three Zn 2þ ions ligated to amino acids and water molecules. ...
... Zn3 is ligated to amino acids Trp1, His14 and Asp122. Zn3 was selected as the centre of binding as it was reported that the Zn 2þ ions play an important role for the catalytic cleavage by binding to charged phosphate groups on the phospholipid substrates [11,17,26]. ...
Phosphatidylcholine-specific phospholipase C (PC-PLC) is a promising target for new anticancer treatment. Herein, we report our work in the discovery of novel drug-like PC-PLC inhibitors. Virtual screening led to the identification of promising hits from four different structural series that contain the molecular scaffold of benzenesulphonamides (10), pyrido[3,4-b]indoles (22), morpholinobenzoic acid (84) and benzamidobenzoic acid (80). 164 structural analogues were tested to investigate the chemical space around the hit series and to generate preliminary structurally activity relationships (SAR). Two of the pyrido[3,4-b]indoles (22_10 and 22_15) had comparable or better potency as D609, an established but non-drug-like PC-PLC inhibitor. Furthermore, three morpholinobenzoic acids (84, 84_4 and 84_5) had superior potency than D609. Therefore, this study paves the way towards the development of drug-like PL-PLC inhibitors as potential anticancer agents.
... Similarly, PLC Bt activity was enhanced with 0.5 mM Mg 2+ , showing a maximum specific activity of 6000 U/mg [2], whereas B. licheniformis PLC required Mg 2+ or Mn 2+ to express its maximal activity [6]. These findings are supported by the crystal structure resolution of PLC Bc , which displays three zinc ions in its active site that are closely associated with residues near to the catalytic site [43]. PLC Bc thus belongs to the metalloenzymes family, possessing a tri-metal zinc center that plays a crucial role in catalytic hydrolysis [44]. ...
... Similarly, PLCBt activity was enhanced with 0.5 mM Mg 2+ , showing a maximum specific activity of 6000 U/mg [2], whereas B. licheniformis PLC required Mg 2+ or Mn 2+ to express its maximal activity [6]. These findings are supported by the crystal structure resolution of PLCBc, which displays three zinc ions in its active site that are closely associated with residues near to the catalytic site [43]. PLCBc thus belongs to the metalloenzymes family, possessing a tri-metal zinc center that plays a crucial role in catalytic hydrolysis [44]. ...
A novel thermoactive phosphatidylcholine-specific phospholipase C (PC-PLCBs) was identified from Bacillus stearothermophilus isolated from a soil sample from an olive oil mill. Enhanced PLCBs production was observed after 10 h of incubation at 55 °C in a culture medium containing 1 mM of Zn2+ with an 8% inoculum size and 6 g/L glucose and 4/L yeast extract as the preferred carbon energy and nitrogen sources, respectively. PLCBs was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and anion exchange chromatography, resulting in a purification factor of 17.6 with 39% recovery. Interestingly, this enzyme showed a high specific activity of 8450 U/mg at pH 8–9 and 60 °C, using phosphatidylcholine PC as the substrate, in the presence of 9 mM sodium deoxycholate and 0.4 mM Zn2+. Remarkable stability at acidic and alkali pH and up to 65 °C was also observed. PLCBs displayed a substrate specificity order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine > sphingomyelin > phosphatidylinositol > cardiolipin and was classified as a PC-PLC. In contrast to phospholipases C previously isolated from Bacillus strains, this PLCBs substrate specificity was correlated to its hemolytic and anti-bacterial potential against erythrocytes and Gram-positive bacterial membranes, which are rich in glycerophospholipids and cardiolipin. An evaluation of PLCBs soybean degumming process efficiency showed that the purified enzyme reduced the phosphorus content to 35 mg/kg and increased the amount of diacylglycerols released, indicating its ability to hydrolyze phospholipids in the crude soybean oil. Collectively, PLCBs could be considered as a potential catalyst for efficient industrial oil degumming, advancing the edible oil industry by reducing the oil gum volume through transforming non-hydratable phospholipids into their hydratable forms, as well as through generating diacylglycerols, which are miscible with triacylglycerols, thereby reducing losses.
... PLC-Y differs in only one amino acid derived from Bacillus cereus PC-PLC. Based on information obtained from the crystal structure of the native enzyme, the phenylalanine at position 66 of the cholinebinding pocket was substituted by a tyrosine (Hansen et al., 1993;Hough et al., 1989). The modified PLC possesses a higher activity toward PE . ...
... Sequence alignment showed that TkPLC presents a low percentage of identity with BcPLC. However, the zinc-binding residues in the BcPLC active site (Hough et al., 1989) are fully conserved in TkPLC. Structural modeling showed that, unlike BcPLC, TkPLC presents a domain inserted between the last two helices of the PLC fold, which we called the CUB-like domain, whose function is still unknown. ...
... The phosphatidylcholine preferring phospholipase C from Bacillus cereus (BcPLC) is a highly characterized phospholipase. The structures of native BcPLC, as well as BcPLC complexes with different ligands, have been solved by x-ray crystallography (Hough et al. 1989). It is a monomeric 28.5 kDa exoenzyme with three zinc ions in its active site and PLCs currently used in enzymatic degumming derive from this enzyme. ...
... This fact is supported by the synteny conservation in their encoding regions (not shown). In BcPLC, there are nine residues known to be involved in binding three zinc ions in its active site: 5 His, 2 Asp, 1 Glu, and 1 Trp (W39, H52, D93, H107, H156, D160, H166, H180, E184) (Hough et al. 1989); all of them are fully conserved according to the alignment presented in Fig. 1a. All identified sequences encode N-terminal secretion signals recognized by the Sec secretion system, as determined by Phobius software (Käll et al. 2007), suggesting that these proteins are secreted. ...
The implementation of cleaner technologies that minimize environmental pollution caused by conventional industrial processes is an increasing global trend. Hence, traditionally used chemicals have been replaced by novel enzymatic alternatives in a wide variety of industrial-scale processes. Enzymatic oil degumming, the first step of the oil refining process, exploits the conversion catalyzed by phospholipases to remove vegetable crude oils’ phospholipids. This enzymatic method reduces the gums’ volume and increases the overall oil yield. A thermostable phospholipase would be highly advantageous for industrial oil degumming as oil treatment at higher temperatures would save energy and increase the recovery of oil by facilitating the mixing and gums removal. A thermostable phosphatidylcholine (PC) (and phosphatidylethanolamine (PE))-specific phospholipase C from Thermococcus kodakarensis (TkPLC) was studied and completely removed PC and PE from crude soybean oil at 80 °C. Due to these characteristics, TkPLC is an interesting promising candidate for industrial-scale enzymatic oil degumming at high temperatures.
Key points
• A thermostable phospholipase C from T. kodakarensis (TkPLC) has been identified.
• TkPLC was recombinantly produced in Pichia pastoris and successfully purified.
• TkPLC completely hydrolyzed PC and PE in soybean oil degumming assays at 80 °C.
... synthesized with a signal sequence on N-terminal (24 amino acid residues) is secreted into the extracellular space in the form of a propeptide. The active form of the enzyme (amino acid residues 245) is formed by cleavage of 14 N-terminal amino acid residues by cellular proteases (Fig. 3b) [33][34][35]. ...
... Tertiary model (a) and secondary (b) structure of phosphatidyl-specific phospholipase C B. cereus (PDB: 1AH7)[33][34][35]. ...
... In Fig. 8 the amino acid sequences of two a-toxins from different strains of C. perfringens are compared with the sequences of two phospholipase C enzymes from different isolates of B. cereus (52,85). The crystal structure of one of the B. cereus enzymes, PC-PLC, has been deduced at a resolution of 0.16 nm (75), and this has been of great value in interpreting the organization of the clostridial a-toxin protein. ...
... The crystallographic structure of PC-PLC (75) shows the enzyme to comprise 10 a-helical segments linked by 1-turns of variable length. Structural predictions (176) suggest a very similar organization for a-toxin, with the additional COOHterminal domain predicted to contain a further four a-helices. ...
Clostridium perfringens is the causative agent of a number of human diseases, such as gas gangrene and food poisoning, and many diseases of animals. Recently significant advances have been made in the development of C. perfringens genetics. Studies on bacteriocin plasmids and conjugative R plasmids have led to the cloning and analysis of many C. perfringens genes and the construction of shuttle plasmids. The relationship of antibiotic resistance genes to similar genes from other bacteria has been elucidated. A detailed physical map of the C. perfringens chromosome has been prepared, and numerous genes have been located on that map. Reproducible transformation methods for the introduction of plasmids into C. perfringens have been developed, and several genes coding for the production of extracellular toxins and enzymes have been cloned. Now that it is possible to freely move genetic information back and forth between C. perfringens and Escherichia coli, it will be possible to apply modern molecular methods to studies on the pathogenesis of C. perfringens infections.
... The first bacterial Zn PC-PLC characterized was the one produced by Bacillus cereus. The initial studies used the enzyme produced by the original strain (Hough et al., 1989;Little & Otnäss, 1975). The gene was later cloned, which allowed heterologous expression in Escherichia coli (Tan et al., 1997). ...
Phospholipase C (PLC) enzymes play a pivotal role in the degumming process of edible oils, which is essential for producing high‐quality refined oils. The enzymatic degumming process, compared with conventional chemical methods, offers significant advantages including improved oil yield, reduced environmental impact, and lower operational costs. However, the industrial application of PLC enzymes is often hindered by their limited stability under harsh processing conditions. This has driven extensive research efforts toward engineering thermostable PLC variants that can withstand the high temperatures and harsh environments typical of oil refining processes. In this review, we explore the latest advancements in the application, design and optimization of thermostable PLC enzymes. We discuss the fundamental economic and ecological interest behind the goal of obtaining thermostable enzymes, and two approaches to the problem, namely the search for enzymes in thermophilic organisms and the design of new sequences with improved stability and activity parameters.
... long prosequence are cleaved to form the 245 a.a. mature enzyme [9]. Although PC-PLC from B. cereus and B. anthracis are successfully used in industry, the creation of improved enzymes is the subject of interest [8]. ...
Microbial enzymes have a leading position among industrial enzymes due to their enormous variety and valuable technological properties. Phospholipases that catalyze the cleavage of phospholipids are the most widely used lipolytic enzymes. In this article, we present development and comparative study of 12 recombinant variants of Phosphatidylcholine-specific phospholipase C which we previously selected from the natural collection of Bacillus thuringiensis as a promising to create industrial producers of enzymes used in the process of plant oil degumming and in other biotechnological areas.
... N-terminal signal sequence to be directed into extracellular space. The active form of the enzyme is formed upon cleavage of the 14 amino acids from N-terminal prosequence by cellular proteases [7]. The mature enzyme is a small monomeric protein of 28 kDa consisting of 245 residues that binds three Zn 2+ ions in its active site [8]. ...
Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to diacylglycerol and phosphate monoesters. It has many applications in the enzymatic degumming of plant oils. Bacterial Phosphatidylcholine-specific PLC from Bacillus spp. is an optimal choice for industrial application in terms of its wide substrate spectrum, thermal stability, high activity, and approved safety. In this work, screening of plc gene sequences of Bacillus thuringiensis isolates, including bacteria collected from the geyser field on the Kamchatka Peninsula, was performed. Moreover, 96 plc gene variants were sequenced and analyzed. Finally, 12 coding sequences for different plc variants were identified. These coding sequences could be considered as promising candidates for the development of producer strains.
... The N-terminal domain (residues 1-246) contains the active site and consists of nine tightly packed alpha-helices [19]. CpPLC contains three essential Zn +2 atoms in its N-terminal domain, as also observed for B. cereus [21] and L. monocytogenes PLC [13], and these could be removed by ethylenediaminetetraacetic acid (EDTA) or o-phenanthroline. Asp56 is a critical residue in the Zn +2 binding site of CpPLC [22], and the D56G substitution changes the secondary structure and abolishes toxicity [23]. ...
Bacterial phospholipases and sphingomyelinases are lipolytic esterases that are structurally and evolutionarily heterogeneous. These enzymes play crucial roles as virulence factors in several human and animal infectious diseases. Some bacterial phospholipases C (PLCs) have both phosphatidylcholinesterase and sphingomyelinase C activities. Among them, Listeria monocytogenes PlcB, Clostridium perfringens PLC, and Pseudomonas aeruginosa PlcH are the most deeply understood. In silico predictions of substrates docking with these three bacterial enzymes provide evidence that they interact with different substrates at the same active site. This review discusses structural aspects, substrate specificity, and the mechanism of action of those bacterial enzymes on target cells and animal infection models to shed light on their roles in pathogenesis.
... The crystal structure of PC-PLC BC was obtained from PDB (ID: 1AH7), resolution of 1.5 Å [31]. ...
Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key enzyme involved in the metabolism of the mammalian phospholipid phosphatidylcholine into secondary messengers diacylglycerol (DAG) and phosphocholine. DAG and phosphocholine have been identified to amplify various cellular processes involved in oncogenesis such as proliferation, cell-cycle activation, differentiation and motility, therefore making PC-PLC a potential target for novel anti-cancer treatments. The current literature standard for PC-PLC inhibition, tricyclodecan-9-yl-potassium xanthate (D609), has been shown to arrest proliferation in multiple cancer cell lines, however, it is not drug-like resulting in low aqueous stability, making it a poor drug candidate. 2-Morpholinobenzoic acids have been shown to have improved PC-PLC inhibitory activity compared to D609, with molecular modelling identifying chelation of the carboxylic acid to catalytic Zn²⁺ ions in the PC-PLC active site being a key interaction. In this study, the carboxylic acid motif was replaced with a hydroxamic acid to strengthen the Zn²⁺ interaction. It was found that the hydroxamic acid derivatives displayed PC-PLC inhibitory activity similar, or better, than D609. Furthermore, these novel inhibitors had potent anti-proliferative activity in MDA-MB-231 and HCT-116 cancer cell lines, far greater than D609 and previous 2-morpholinobenzoic acids.
... 18,19 It is a monomeric enzyme with a tri-metallic zinc centre at the active site. 20,21 In B. cereus, PC-PLC Bc is translated as a preproprotein, which subsequently is processed into its mature form (Table S1 and Fig. S1 †). 22,23 Mature PC-PLC Bc is secreted by the bacteria as an exoenzyme. ...
The Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLCBc) is an enzyme that catalyses the hydrolysis of phosphatidylcholines into phosphocholine and 1,2-diacylglycerols. PC-PLCBc has found applications in both the food industry and in medicinal chemistry. Herein, we report our work in the development and optimisation of a matrix assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry-based assay to monitor PC-PLCBc activity. The use of one-phase and two-phase reaction systems to assess the inhibition of PC-PLCBc with different structural classes of inhibitors was compared. We also highlighted the advantage of our assay over the commonly used commercially available Amplex Red assay. This method will also be applicable to work on the activity and inhibition of other phospholipases.
... Zinc clusters have been observed in the active sites of many enzymes [20] . Seven structures that contain at least two zinc ions have been examined: alkaline phosphatase [21] , phosphotriesterase [22] , Klenow fragment of DNA polymerase I [23] , P1 nuclease [24] , phospholipase C [25] , aminopeptidase [26] , and leucine aminopeptidase [27] . However, the enzyme from Bacillus cereus functioned reasonably well with only one bound zinc ion [28] . ...
Chlorothalonil hydrolytic dehalogenase (Chd) is one of two reported hydrolytic dehalogenases for halogenated aromatics, and its catalysis is independent of coenzyme A and ATP. Earlier studies have established that the catalytic activity of Chd requires zinc ions. In this study, the metal center of Chd was systematically investigated. The metal content of Chd was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES), and there were 2.14 equivalents of zinc/mol of protein, indicating that Chd contains a binuclear (Zn2+-Zn2+) center. It was found that other divalent cations, such as cobalt (Co2+) and cadmium (Cd2+), could substitute zinc (Zn2+) leading to relative activities of 91.6% and 120.0%, whereas manganese (Mn2+) and calcium (Ca2+) could substitute Zn2+ leading to relative activities of 29.1% and 57.0%, respectively. The enzymatic properties of these different metal ion-substituted Chd variants were also compared. Error-prone PCR and DNA shuffling methods were applied to directly evolve Chd to generate variants with higher catalytic efficiencies of chlorothalonil. Enhanced Chd variants were selected based on the formation of clear haloes on Luria-Bertani plates supplemented with chlorothalonil. One variant, Q146R/N168Y/S303G, exhibited a 4.43-fold increase in catalytic efficiency, showing the potential for application in the dehalogenation and detoxification of chlorothalonil contaminated-sites.
... Studies also reported the production of Cry toxins in other bacteria like Bacillus popilliae and Clostridium bifermentans [13]. [107]), (C) Vip2 toxin (adopted from Chakroun et al. [19]), (D) phospholipase C (adopted from Hough et al. [108]), (E) ChiA from Serritia marcescens, (F) ChiB from Serritia marcescens (adopted from Horn et al. [109]), (G) hirsutellin (adopted from Olombrada et al. [51]), (H) Yersinia entomophaga toxin complex. (Adopted from Landsberg et al. [110]) Single gene-derived toxins, target specificity, risk-free against humans, nontargets and beneficials, biodegradability, etc. are the major characteristic features governing its wide usage. ...
... Also it is shown that some hydrolytic enzymes have the ability to cleavage the phosphate ester bonds. In the active sites of such enzymes, two or more transition metal ions are located which cooperatively operate as Lewis acid sites in the catalytic process [22][23][24][25][26]. ...
In this paper, in vitro DNA binding of a water-soluble metal–organic coordination polymer of Zn(II) Schiff base complex was considered. By focusing on the cooperative effect of multi-Zn(II) centers of the complex, the affinity and mode of binding were investigated by some techniques, e.g., absorption and fluorescence spectroscopy, circular dichroism spectroscopy, viscosity measurement, molecular docking, iodide quenching studies and effect of ionic strength on the complex–DNA binding. The calculated value of Kb = 1.3 × 10³ M⁻¹ represented a good affinity of the Zn(II) complex to DNA. From the experimental methods and docking studies, non-classical intercalation (i.e., van der Waals interactions, groove binding or external binding) was proposed as the main mode of binding.
... When these histidines are replaced by other amino acid residues, such as glycine, the hemolytic activity and lethality of the α-toxin are significantly reduced or even eliminated. Nonetheless, its antigenicity can be retained [12][13][14], pointing to a promising strategy for the development of a subunit vaccine against C. perfringens α-toxin [15,16]. ...
Clostridium perfringens α-toxin is one of the major virulence factors during C. perfringens infection, causing hemolysis of erythrocytes in various species. Here, genetically engineered Lactobacillus casei (pPG-α/L. casei 393) constitutively expressing the toxoid of C. perfringens α-toxin was generated and its immunogenicity in mice for induction of protective immunity against the α-toxin was evaluated via oral immunization. The α-toxoid was constitutively expressed by pPG-α/L. casei 393 without a specific inducer, as confirmed by western blotting, laser confocal microscopy, and flow cytometry. In an experiment on BALB/c mice to evaluate the oral immunogenicity of pPG-α/L. casei 393, significant levels of a specific secretory IgA (sIgA) antibody in the intestinal mucus and feces and an IgG antibody in the serum of the probiotic vaccine group were detected after booster immunization (p < 0.05) as compared with the pPG/L. casei 393 and PBS control groups. These antibodies effectively neutralized C. perfringens natural α-toxin. Moreover, significantly higher levels of cytokines IL-2, IL-4, IL-10, IL-12, IL-17, and interferon (IFN) γ in the serum and increased proliferation of spleen lymphocytes obtained from mice orally immunized with pPG-α/L. casei 393 were detected. With a commercial C. perfringens type A inactivated vaccine as a control, immune protection provided by the probiotic vaccine against C. perfringens α-toxin was evaluated, and 90% and 80% protection rates were observed, respectively. Therefore, strain pPG-α/L. casei 393 effectively elicited mucosal, humoral, and cellular immunity, suggesting that pPG-α/L. casei 393 is a promising candidate for development of a vaccine against C. perfringens α-toxin.
... PLC-Y, derived from Bacillus cereus PC-PLC, possesses a point mutation that replaces the natural phenylalanine at position 66 with tyrosine. The crystal structure of the native protein indicates that position 66 is part of the choline binding pocket (Hansen et al. 1993;Hough et al. 1989). This mutation of the amino acid at position 66 makes the protein more selective for PE than the wild type PC-PLC enzyme. ...
Phospholipids play a central role in all living organisms. Phospholipases, the enzymes aimed at modifying phospholipids, are consequently widespread in nature and play diverse roles, from lipid metabolism and cellular signaling in eukaryotes to virulence and nutrient acquisition in microbes. Phospholipases catalyze the hydrolysis of one or more ester or phosphodiester bonds of glycerophospholipids. The use of phospholipases with industrial purposes has constantly increased over the last 30 years. This demand is rapidly growing given the ongoing improvements in protein engineering and the reduction of enzymes manufacturing costs, making them suitable for industrial use. Here, a general overview of phopholipases A, B, C, and D and their industrial application is presented along with potential new uses for these enzymes. We draw attention to commercial phospholipases used to improve the emulsifying properties of products in the baking, egg, and dairy industries. On the other hand, the improvement of oil degumming by phospholipases is thoroughly analyzed. Moreover, recent developments in enzymatic biodiesel production and the use of phospholipases for the synthesis of phospholipids with pharmaceutical or nutritional value are reviewed.
... Studies also reported the production of Cry toxins in other bacteria like Bacillus popilliae and Clostridium bifermentans [13]. [107]), (C) Vip2 toxin (adopted from Chakroun et al. [19]), (D) phospholipase C (adopted from Hough et al. [108]), (E) ChiA from Serritia marcescens, (F) ChiB from Serritia marcescens (adopted from Horn et al. [109]), (G) hirsutellin (adopted from Olombrada et al. [51]), (H) Yersinia entomophaga toxin complex. (Adopted from Landsberg et al. [110]) Single gene-derived toxins, target specificity, risk-free against humans, nontargets and beneficials, biodegradability, etc. are the major characteristic features governing its wide usage. ...
... Its synthesis was achieved through condensation of tren with excess 2-picolylchloride hydrochloride. Reaction of the tren-bpma ligand with Cu(NO 3 ) 2 Á2.5H 2 O afforded a tris-copper(II) complex [22]; (B) phospholipase C from Bacillus cereus (PDB 1AH7) [23]. Zn ions are colored in black and P atoms in yellow. ...
Tris-copper centers are present at the active site of multicopper oxidases (MCO) which couple the four-electron reduction of molecular oxygen to water with the oxidation of substrates. Modelling these sites with small molecular complexes has thus attracted the interest of many groups in the (bio)inorganic community over the past three decades and still appears a challenge. These enzymes and their model complexes presently enjoy a renewed interest as potential non-precious metal catalyst for the Oxygen Reduction Reaction. Moreover recent work has revealed that tris-copper centers can catalyze methane oxidation. Therefore the elaboration of tris-copper system constitutes an important and timely issue. The aim of the present review is to analyze the various attempts at preparing tris-copper complexes in terms of strategy of ligand design. Of course the challenge to be met is to force three independent binding sites to converge and react in concert. Three main approaches have been developed to anchor these binding sites based on the use of (i) a node either (a) a single atom node (tren-based systems and related), or (b) an hexa-atom node (mesityl-based systems and derivatives), (ii) macrocyclic systems, and (iii) combination of mono- and dinuclear sites. The structures of the different systems will be described and analyzed accordingly. Then the various reactivities exhibited by these systems will be presented so as to evaluate how the ligand design influences the reactivity and to discern promising future directions.
Dysregulation of choline phospholipid metabolism and overexpression of phosphatidylcholine-specific phospholipase C (PC-PLC) is implicated in various cancers. Current known enzyme inhibitors include compounds based on a 2-morpholino-5-N-benzylamino benzoic acid, or...
The reactions of [Zn3Cl2(3,5-Me2PzH)4(t-BuPO3)2] with organostibonic acid in varying reaction conditions have been investigated. Single-crystal X-ray diffraction studies reveal the formation of [Zn2(p-ClC6H4Sb)2(O)2(OCH3)2(t-BuPO3)3(py)2] (1), [Zn2(p-ClC6H4SbV)4(SbIII)2(O)8(t-BuPO3H)4(t-BuPO3)2(py)2Cl2] (2), and [Zn2(RSb)4(O)4(OCH3)4(t-BuPO3)4(py)2], where R = p-ClC6H4 (3) and R = p-iPrC6H4 (4), respectively. Interestingly, in the synthesis of 2, complete dearylation of organoantimony moieties followed by C-F bond formation, a reduction from Sb (V) to Sb (III), and Sb···Cl weak intermolecular interactions have been observed. ESI-MS studies suggested that clusters 1-4 maintained their structural integrity in the solution state also. Solution NMR studies (1H, 31P, and 13C) support well the observed solid-state structures. 1-4 were tested for antibacterial activity using a microdilution assay. 1 and 4 showed the best activity with lower MIC values (0.78-6.25 μg/mL) against all the tested pathogens. The total antioxidant activity of 1-4 was evaluated through the phosphomolybdenum assay, which showed a total antioxidant activity ranging from 28.96 to 86.46 mg AAE/g compound with the ascorbic acid standard.
Computational methods in medicinal chemistry facilitate drug discovery and design. In particular, machine learning methodologies have recently gained increasing attention. This chapter provides a structured overview of the current state of computational chemistry and its applications for the interrogation of the endocannabinoid system (ECS), highlighting methods in structure-based drug design, virtual screening, ligand-based quantitative structure–activity relationship (QSAR) modeling, and de novo molecular design. We emphasize emerging methods in machine learning and anticipate a forecast of future opportunities of computational medicinal chemistry for the ECS.Key wordsEndocannabinoid SystemComputational ChemistryMachine LearningStructure-Based Drug DesignVirtual ScreeningQSARDe Novo Drug Design
A still unsolved, although critical, issue in endocannabinoid research is the mechanism by which the lipophilic anandamide (AEA) moves from its site of synthesis, crosses the aqueous milieu, and reaches the different intracellular membrane compartments, where its metabolic and signaling pathways take place. The difficulty of studying intracellular AEA transport and distribution results from the lack of specific probes and techniques to track and visualize this bioactive lipid within the cells. Herein, we describe the use of a biotinylated, non-hydrolyzable derivative of AEA (biotin-AEA, b-AEA) for visualizing the subcellular distribution of this endocannabinoid by means of confocal fluorescence microscopy.Key wordsEndocannabinoidsAnandamideSubcellular distributionGlass coverslipsBiotinylated derivative of anandamideConfocal microscopy
Extraction and quantification of endocannabinoids from biological tissues is essential to unravel their changes under physiological and pathophysiological conditions. We describe here an analytical protocol for the extraction of endocannabinoids, anandamide (archidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and endocannabinoid-like lipids such as palmitoyl ethanolamide (PEA) and oleoyl ethanolamide (OEA), as well as arachidonic acid (AA) from biological tissues using liquid–liquid extraction method and simultaneous quantification by liquid chromatography multiple reaction monitoring (LC/MRM).Key wordsEndocannabinoidsEndocannabinoid-like lipidsLiquid-chromatographyMultiple reaction monitoringLipid extractionLipidomics
Phospholipase C (PLC) represents an important type of enzymes with the feature of hydrolyzing phospholipids at the position of the glycerophosphate bond, among which PLC extracted from Bacillus cereus (BC-PLC) has been extensively studied owing to its similarity to hitherto poorly characterized mammalian analogues. This study focuses on investigating the interfacial hydrolysis mechanism of phosphatidylcholine (PC) monolayer and bilayer membranes catalyzed by BC-PLC using sum frequency generation vibrational spectroscopy (SFG-VS) and laser scanning confocal microscopy (LSCM). We found that, upon interfacial hydrolysis, BC-PLC was adsorbed onto the lipid interface and catalyzed the lipolysis with no net orientation, as evidenced by the silent amide I band, indicating that ordered PLC alignment was not a prerequisite for the enzyme activity, which is very different from what we have reported for phospholipase A1 (PLA1) and phospholipase A2 (PLA2) [Kai, S. Phys. Chem. Chem. Phys. 2018, 20(1), 63-67; Wang, F. Langmuir 2019, 35(39), 12831-12838; Zhang, F. Langmuir 2020, 36(11), 2946-2953]. For the PC monolayer, one of the two hydrolysates, phosphocholine, desorbed from the interface into the aqueous phase, while the other one, diacylglycerol (DG), stayed well packed with high order at the interface. For the PC bilayer, phosphocholine dispersed into the aqueous phase too, similar to the monolayer case; however, DG, presumably formed clusters with the unreacted lipid substrates and desorbed from the interface. With respect to both the monolayer and bilayer cases, mechanistic schematics were presented to illustrate the different interfacial hydrolysis processes. Therefore, this model experimental study in vitro provides significant molecular-level insights and contributes necessary knowledge to reveal the lipolysis kinetics with respect to PLC and lipid membranes with monolayer and bilayer structures.
A novel alkaline thermostable phosphatidylcholine-specific phospholipase C (PC-PLCBt) was expressed in E. coli system. Recombinant PC-PLCBt (rPC-PLCBt) activity and thermostability were shown to be significantly dependent on the Zn²⁺. The maximum rPC-PLCBt catalytic activity was found to be 1372 U mg⁻¹ in the presence of 0.1 mM Zn²⁺ and at 60 °C using an Egg PC as substrate. The interfacial kinetic data show that nPC-PLCBt and rPC-PLCBt display similar substrate specificity on various phospholipid monolayers. The maximal rPC-PLCBt activities were recorded, at decreasing order, on 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-diacyl-sn-phosphoglycerol (PG), and 1,2-diacyl-sn-phosphoserine (PS) monolayers at interfacial surface pressures of 15, 25, 20, and 25 mN m⁻¹, respectively. Such important penetrating power could be exploited for pharmacological purposes. The highest activities were recorded on the DLPC monolayer and shown to be 121.61 and 40.13 mmol cm⁻² min⁻¹ M⁻¹ for native and recombinant PC-PLCBt, respectively. Interestingly, compared to all known Bacillus PLCs, both PC-PLCBt forms showed an exclusive capacity to hydrolyze the PG film with a more pronounced rate of hydrolysis for the native form with a specific activity of 58.29 mmol cm⁻² min⁻¹ M⁻¹. Therefore, the high enzyme level production of about 14 mg L⁻¹, the thermostability as well as the broad phospholipid specificity of PC-PLCBt represent great potential in the crude oil refining industry.
In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.
Listeria monocytogenes is an intracellular food-borne pathogen that causes listeriosis, a severe and potentially life-threatening disease. Listeria uses a number of virulence factors to proliferate and spread to various cells and tissues. In this process, three bacterial virulence factors, the pore-forming protein listeriolysin O and phospholipases PlcA and PlcB, play a crucial role. Listeriolysin O belongs to a family of cholesterol-dependent cytolysins that are mostly expressed by gram-positive bacteria. Its unique structural features in an otherwise conserved three-dimensional fold, such as the acidic triad and proline-glutamate-serine-threonine-like sequence, enable the regulation of its intracellular activity as well as distinct extracellular functions. The stability of listeriolysin O is pH- and temperature-dependent, and this provides another layer of control of its activity in cells. Moreover, many recent studies have demonstrated a unique mechanism of pore formation by listeriolysin O, i.e., the formation of arc-shaped oligomers that can subsequently fuse to form membrane defects of various shapes and sizes. During listerial invasion of host cells, these membrane defects can disrupt phagosome membranes, allowing bacteria to escape into the cytosol and rapidly multiply. The activity of listeriolysin O is profoundly dependent on the amount and accessibility of cholesterol in the lipid membrane, which can be modulated by the phospholipase PlcB. All these prominent features of listeriolysin O play a role during different stages of the L. monocytogenes life cycle by promoting the proliferation of the pathogen while mitigating excessive damage to its replicative niche in the cytosol of the host cell.
The actinomycete Amycolatopsis japonicum is the producer of the chelating compound [S,S]-ethylenediamine-disuccinc acid (EDDS). [S,S]-EDDS is an isomer of ethylenediamine-tetraacetic acid (EDTA), an economically important chelating compound that suffers from an extremely poor degradability. Frequent use of the persistent EDTA in various industrial and domestic applications has caused an accumulation of EDTA in soil as well as in aqueous environments. As a consequence, EDTA is the highest concentrated anthropogenic compound present in water reservoirs. The [S,S]-form of EDDS has chelating properties similar to EDTA, however, in contrast to EDTA it is readily biodegradable. In order to compete with the cost-effective chemical synthesis of EDTA, we aimed to optimize the biotechnological production of [S,S]-EDDS in A. japonicum by using metabolic engineering approaches. Firstly, we integrated several copies of the [S,S]-EDDS biosynthetic genes into the chromosome of A. japonicum and replaced the native zinc responsive promoter with the strong synthetic constitutive promoter SP44*. Secondly, we increased the supply of O-phospho-serine, the direct precursor of [S,S]-EDDS. The combination of these approaches together with the optimized fermentation process led to a significant improvement in [S,S]-EDDS up to 9.8 g/L with a production rate of 4.3 mg/h/g DCW.
Two Zinc(II) complexes [Zn4(L1)4 ]·2H2O (1) and [Zn2(L2)2]·2H2O (2) of pyruvaldehydethiosemicarbazone ligands are reported. The complexes were characterized by elemental analysis, IR, NMR, UV-vis spectroscopy and by single-crystal X-ray crystallography. X-ray crystal structure determinations of the complexes show that though Zn : ligand stoichiometry is 1 : 1 in both the complexes, the molecular unit is tetranuclear for 1 and binuclear for 2. Both the complexes show selective sensing of ATP at pH 7.4 (0.01 M HEPES) in CH3CN-H2O (9 : 1) medium in the presence of other anions like AcO(-), NO3(-), F(-), Cl(-), H2PO4(-) , HPO4(2-) and P2O7(2-). The UV-titration experiments of complexes 1 and 2 with ATP results in binding constants of 2.0(+-E0.07) x 10^4 M-1 and 7.1(+-E0.05) x 10^3 M-1 respectively. The calculated detection limits of 6.7 mM and 1.7 mM for 1 and 2 respectively suggest that the complexes are sensitive detectors of ATP. High selectivity of the complexes is confirmed by the addition of ATP in presence of an excess of other anions. DFT studies confirm that the ATP complexes are more favorable than those with the other inorganic phosphate anions, in agreement with the experimental results. Phosphatase like activity of both complexes is investigated spectrophotometrically using 4-nitrophenylphosphate (NPP) as a substrate, indicating the complexes possess significant phosphate ester hydrolytic efficiency. The kinetics for the hydrolysis of the substrate NPP was studied by the initial rate method at 25 C. Michaelis-Menten derived kinetic parameters indicate that rate of hydrolysis of the P-O bond by complex 1 is much greater than that of complex 2, the kcat values being 212(+-5) and 38(+-2) h-1 respectively. The DNA binding studies of the complexes were investigated using electronic absorption spectroscopy and fluorescence quenching. The absorption spectral titrations of the complexes with DNA indicate that the CT-DNA binding affinity (Kb) of complex 1 (2.10(+-0.07) x 10^6 M -1) is slightly greater than that of 2 (1.11(+-0.04) x 10^6 M-1). From fluorescence spectra the apparent binding constant (Kapp) values were calculated and they are found to be 5.41(+-0.01) x 10^5 M-1 for 1 and 3.93(+-0.02) x 10^5 M-1 for 2. The molecular dynamics simulation demonstrates that the Zn(II) complex 1 is a good intercalator of DNA.
An avirulent pleiotropic mutant of the insect pathogen Bacillus thuringiensis subsp. gelechiae, isolated by Heierson et al. (A. Heierson, I. Sidén, A. Kivaisi, and H. G. Boman, J. Bacteriol. 167:18-24, 1986) as a spontaneous phage-resistant mutant, was further characterized and found to lack the expression of phosphatidylcholine- and phosphatidylinositol-degrading phospholipase C, beta-lactamase, and flagellin because of the absence of corresponding mRNAs. The avirulent mutant was also found to be less efficient in killing insect cells in vitro than the wild type and to have altered behavior in vivo; wild-type B. thuringiensis does not circulate in the insect hemolymph after injection, whereas the avirulent mutant and nonpathogenic control bacteria remain in circulation. Flagella and motility may be important for virulence in the early stages of an infection; mutants with decreased motility appear less virulent when fed to Trichoplusia ni but not when injected. The 50% lethal doses of wild-type strain Bt13 and avirulent mutant strain Bt1302 were estimated to be 0.52 +/- 0.25 and 2,600 +/- 1,300 CFU per injected larva, respectively.
Histotoxic Clostridial Infections, Page 1 of 2
Abstract
The pathogenesis of clostridial myonecrosis or gas gangrene involves an interruption to the blood supply to the infected tissues, often via a traumatic wound, anaerobic growth of the infecting clostridial cells, the production of extracellular toxins, and toxin-mediated cell and tissue damage. This review focuses on host-pathogen interactions in Clostridium perfringens-mediated and Clostridium septicum-mediated myonecrosis. The major toxins involved are C. perfringens α-toxin, which has phospholipase C and sphingomyelinase activity, and C. septicum α-toxin, a β-pore-forming toxin that belongs to the aerolysin family. Although these toxins are cytotoxic, their effects on host cells are quite complex, with a range of intracellular cell signaling pathways induced by their action on host cell membranes.
A new macroacyclic Schiff base, 1,3-bis(2((Z)-(hydroxyethylimino)methyl) phenoxy)propan-2-ol, (H3L), was synthesized and characterized by infrared, ultraviolet–visible and NMR spectroscopies, and microanalysis. A series of new complexes was subsequently obtained from the reaction of the H3L ligand and Cd(II), Zn(II) and Cu(II). The prepared complexes [Cd2H2L(H2O)(NO3)2]NO3 (1), [ZnH2L]ClO4 (2), and [CuH2L]ClO4 (3), were characterized by IR spectroscopy and microanalysis, and, in the case of 1 and 2, also by NMR spectroscopy. Suitable crystals of 1 were obtained for X-ray analysis. The single crystal X-ray structural analysis of 1 showed that the complex is binuclear, with all nitrogen and oxygen atoms of the ligand (N2O5) coordinated to the two Cd(II) ions. In the crystal structure the two distorted mono-capped anti-prism Cd(II) centers are bridged asymmetrically by an oxygen of a hydroxyl group of the Schiff base and a water molecule. Density functional theory calculations were performed to characterize the conformational equilibrium of the Schiff base in solution. The geometry and the ¹³C NMR spectrum of the Cd(II) complex were also calculated by DFT, confirming the presence of the complex in the investigated solution media. Time dependent-DFT calculations provided further insight on the interpretation of the experimental UV–Vis spectrum of the Cd(II) complex.
The isolated Tunisian strain of Bacillus thuringiensis was characterized by the secretion of a phospholipase C (PLC). The activity of the extracellular PLC from B. thuringiensis (PLC Bt ) was optimized and the enzyme was purified to homogeneity. In fact, the yield reached 50% and the apparent molecular mass was determined to be 28 kDa as analyzed by SDS-PAGE and the N-terminal sequence PLC Bt was characterized by a high degree of homology with those of other Bacillus PLC. Herein, we report for the first time a thermoactive PLC with a higher specific activity of about 6000 U/mg measured at 55–60 °C and pH 7–8 using phosphatidylcholine as a substrate in presence of 0.5 mM Mg ²⁺ . PLC Bt retains more than half of its maximal activity between 30–45 °C at pH 7–8.5 and tolerates pH values higher than 11. The PLC Bt substrate specificity showed the following decreasing order in vitro: phosphatidylcholine >> phosphatidylethanolamine > phosphatidylserine ≥ phosphatidylglycerol; while phosphatidic acid and phosphatidylinositol were poor substrates. The PLC Bt , with its biochemical properties, could be considered as a potential material for industrial and biotechnological applications. Among them, the production of emulsifiers and industrial degumming purposes either for the edible oil industry.
Two tripodal hexa-urea receptors functionalized with aromatic terminal groups are capable of binding choline phosphate (CP). Crystal structures of the host-guest complexes reveal that the zwitterion CP is efficiently encapsulated in the tripodal hosts by a dual-site binding mode. The phosphate tail of CP is coordinated by the urea groups and the quaternary ammonium head is bound in a ‘composite aromatic box’ through cation∙∙∙π and hydrogen-bonding interactions. Such a partial aromatic binding environment for the Me3N-+ cation mimics that of most enzymes catalyzing the conversion of quaternary ammonium substrates. Moreover, NMR, ESI-MS, and fluorescence studies demonstrate selective binding and sensing of CP over other competing species such as ADP, ATP, choline and derivatives.
Phospholipases are lipolytic enzymes that hydrolyze phospholipid substrates at specific ester bonds. Phospholipases are widespread in nature and play very diverse roles from aggression in snake venom to signal transduction, lipid mediator production, and metabolite digestion in humans. Phospholipases vary considerably in structure, function, regulation, and mode of action. Tremendous advances in understanding the structure and function of phospholipases have occurred in the last decades. This introductory chapter is aimed at providing a general framework of the current understanding of phospholipases and a discussion of their mechanisms of action and emerging biological functions.
To mimic the active sites of the hydrolytic enzyme zinc phosphotriesterase, a new dinucleating unsymmetric ligand, PICIMP (2‐{[2‐hydroxy‐5‐methyl‐3‐({[(1‐methyl‐1H‐imidazol‐2‐yl)methyl](pyridin‐2‐ylmethyl)amino}methyl)benzyl][(1‐methyl‐1H‐imidazol‐2‐yl)methyl]amino}acetic acid), has been synthesized and characterized. The hydrolytic efficacy of the complex solution (PICIMP/ZnCl2 = 1:2) has been investigated using bis‐(2,4‐dinitrophenyl)phosphate (BDNPP), a DNA analogue substrate. Speciation studies were undertaken by potentiometric titrations at varying pH for both the ligand and the corresponding dizinc complex to elucidate the formation of the active hydrolysis catalyst; these studies reveal that the dinuclear zinc(II) complexes, [Zn2(PICIMP)]2+ and [Zn2(PICIMP)(OH)]+ predominate in solution above pH 4. The obtained pKa of 7.44 for the deprotonation of water suggests formation of a bridging hydroxide between the two ZnII ions. Kinetic investigations of BDNPP hydrolysis over the pH range 5.5–10.5 have been performed. The cumulative results indicate the hydroxo‐bridged dinuclear ZnII complex [Zn2(PICIMP)(µ‐OH)]+ as the effective catalyst. Density functional theory calculations were performed to investigate the detailed reaction mechanism. The calculations suggest that the bridging hydroxide becomes terminally coordinated to one of the zinc ions before performing the nucleophilic attack in the reaction. A dinuclear zinc complex with an unsymmetric ligand environment has been prepared and characterized with the aim to make a structural and functional model for the zinc phosphotriesterase active site. Catalytic efficiency of this complex has been investigated by using the phosphodiester BDNPP as a model substrate. Cumulative results from catalytic activity and speciation studies point towards a monohydroxo dinuclear zinc complex as the most effective catalyst, and a likely reaction mechanism has been modelled by DFT calculations.
The 2.5-A crystal structure of the calcium-free form of the dimeric venom phospholipase A2 from the Western Diamondback rattlesnake Crotalus atrox, has been refined to an R-factor of 17.8% (I greater than 2 sigma) and acceptable stereochemistry. The molecule is a nearly perfect 2-fold symmetric dimer in which most of the catalytic residues of both subunits face an internal cavity. The restricted access to the putative catalytic sites is especially puzzling as the optimal substrates for this and most other phospholipase A2 are phospholipids condensed in micellar or lamellar aggregates. We point out that substrate access to the internal cavity may be aided by calcium binding which can alter the intersubunit contacts that shield the catalytic network. We also suggest that a system of hydrogen-bonded moieties exists on the surface of the dimer that links the amino terminus to the catalytic system, through an invariant Gln 4 side chain and the backbone of the active center residue, Tyr 73. This hydrogen-bonded network is on a highly accessible surface of the dimer and would appear to contribute to the enzyme's (as opposed to the proenzyme's) special capacity to attack aggregated rather than monomeric substrate.
The refined high resolution crystal structure of the bovine phospholipase A2 was compared with its counterpart from the venom of Crotalus atrox, the western diamondbacked rattlesnake. The strong similarity in their backbone conformations forms the basis of a common numbering system for the amino acid sequence. The three common major helices and much of the extended chain form a nearly identical “homologous core” structure. The variations in conformation usually arise from deletions/insertions or en bloc shifts of structural units. The exception to this is part of the highly conserved calcium-binding loop; however, this is to be expected as 1) there is no calcium ion sequestered in the venom dimer as there is in the case of the bovine enzyme and 2) two side chains in that segment form dimer-stabilizing interactions between the subunits of the C. atrox enzyme. The absolutely conserved catalytic network of hydrogen-bonded side chains formed by His 48, Tyr 52, Tyr 73, and Asp 99, as well as the hydrophobic wall that shields it, are virtually superimposable in the two structures. However, the details of the structural relationship between the amino terminus and the catalytic network differ in the two species and the ordered water molecules thought to be either functionally or structurally important in the pancreatic enzymes are not found in the crystal structure of the phospholipase A2 from C. atrox. The most striking difference from a functional stand-point is the fact that the surface depression in the region of the catalytic network that has been commonly considered the active site is shielded substantially in forming the intersubunit contact surface of the dimeric venom enzyme.
A simple diagrammatic representation has been used to show the arrangement of alpha helices and beta sheets in 31 globular proteins, which are classified into four clearly separated classes. The observed arrangements are significantly non-random in that pieces of secondary structure adjacent in sequence along the polypeptide chain are also often in contact in three dimensions.
The phospholipase C gene from Clostridium perfringens was isolated, and its sequence was determined. It was found that the structural gene codes for a protein of 399 amino acid residues. The NH2-terminal residues have the typical features of a signal peptide and are probably cleaved after secretion. Escherichia coli cells harboring the phospholipase C gene-containing plasmid expressed high levels of this protein in the periplasmic space. Phospholipase C purified from E. coli transformants was enzymatically active, hemolytic to erythrocytes, and toxic to animals when injected intravenously. The phospholipase C gene from a related organism, Clostridium bifermentans, was also isolated. The two phospholipase C genes were found to be 64% homologous in coding sequence. The C. bifermentans protein, however, was 50-fold less active enzymatically than the C. perfringens enzyme.
The classic pathway for agonist-induced generation of diacylglycerol is via activation of a phospholipase C-mediated hydrolysis of the "phosphoinositides." We now report findings from a variety of cell types, which indicate that tumor-promoting phorbol diesters, serum, and platelet-derived growth factor activate within seconds the hydrolysis of phosphatidylcholine, as detected by the formation of diacylglycerol and phosphocholine. It is known that phorbol diesters do not stimulate hydrolysis of the phosphoinositides. Yet, in cells prelabeled with either [14C]oleate or [32P]orthophosphate, addition of the tumor promoter phorbol dibutyrate (PBt2) resulted in the rapid generation of both diacylglycerol and phosphatidate in a time- and dose-dependent manner. The fatty acid composition of the phosphatidate most resembled the fatty acid profile of phosphatidylcholine from the same cell type. Taken together, these findings suggested a role for protein kinase C in the generation of diacylglycerol (and phosphatidate) from phosphatidylcholine. To define further the pathways involved, the metabolism of cellular phosphatidylcholine was studied. In cells prelabeled with [3H]choline, addition of PBt2, but not 4 alpha-phorbol, stimulated the formation of intracellular phosphocholine within 45 sec. Furthermore, addition of platelet-derived growth factor (PDGF) or serum to "serum-starved" cells prelabeled with [3H]choline resulted in increased levels of intracellular phosphocholine within 15-30 sec. Thus, the data suggest that agonists that stimulate protein kinase C either directly (e.g., PBt2) or indirectly via activation of phosphoinositide hydrolysis (e.g., PDGF and serum) may stimulate degradation of phosphatidylcholine by phospholipase C in intact cells. However, prior down-regulation of protein kinase C by prolonged pretreatment of cells with PBt2 almost totally abolished subsequent stimulation of phosphatidylcholine degradation by PBt2 but only partially attenuated subsequent stimulation by PDGF and serum. These observations suggest that PDGF and serum act, at least partially, through a protein kinase C-independent mechanism. Lastly, the size of the cellular choline and CDP-choline pools were shown to be small and relatively insensitive to agonist addition, as compared to the size and behavior of the phosphocholine pool. Thus, the rapidly increased levels of phosphocholine (and diacylglycerol) arising in response to agonist addition appear to be derived directly from phosphatidylcholine by a phospholipase C-mediated mechanism.
Antibodies against phospholipase C were prepared in rabbits and used to affinity purify a phosphatidylcholine-preferring phospholipase C from a human monocytic cell line. Affinity chromatography resulted in an approximately 3000-fold, one-step enrichment of phospholipase C. The human enzyme had an apparent molecular mass of 40,000 daltons as determined by SDS gel electrophoresis. Western blotting analysis demonstrated that this protein interacted specifically with the rabbit antibody raised against bacterial phospholipase C. The purified enzyme preferred phosphatidylcholine as a substrate, was neutral pH active and was inhibited by EGTA. These studies demonstrate that antibodies raised against bacterial phospholipase C may be useful in purifying phospholipase C from a human source.
The Klenow fragment of E. coli DNA polymerase I has two separate structural domains. The polymerase active site is located on the larger C-terminal domain, about 30 Å from the 3′–5′ exonuclease active site on the N-terminal domain. We are using combination of X-ray crystallography and molecular genetics to investigate the mechanisms of both reactions and the way that these two distant active sites can work together to enhance the fidelity of DNA synthesis.
The crystal structure of bovine pancreatic phospholipase A2 has been refined to 1.7 Å resolution. The starting model for this refinement was the previously published structure at a resolution of 2.4 Å (Dijkstra et al., 1978). This model was adjusted to the multiple isomorphous replacement map with Diamond's real space refinement program (Diamond, 1971,1974) and subsequently refined using Agarwal's least-squares method (Agarwal, 1978). The final crystallographic R-factor is 17.1% and the estimated root-mean-square error in the positional parameters is 0.12 Å. The refined model allowed a detailed survey of the hydrogen-bonding pattern in the molecule. The essential calcium ion is located in the active site and is stabilized by one carboxyl group as well as by a peptide loop with many residues unvaried in all known phospholipase A2 sequences. Five of the oxygen ligands octahedrally surround the ion. The sixth octahedral position is shared between one of the carboxylate oxygens of Asp49 and a water molecule. The entrance to the active site is surrounded by residues involved in the binding of micelle substrates. The N-terminal region plays an important role here. Its α-NH+3 group is buried and interacts with Gln4, the carbonyl oxygen of Asn71 and a fully enclosed water molecule, which provides a link between the N terminus and several active site residues. A total of 106 water molecules was located in the final structure, most of them in a two-layer shell around the protein molecule. The mobility in the structure was derived from the individual atomic temperature factors. Minimum mobility is found for the main chain atoms in the central part of the two long α-helices. The active site is rather rigid.
The structures of pig heart and chicken heart citrate synthase have been determined by multiple isomorphous replacement and restrained crystallographic refinement for two crystal forms, a tetragonal form at 2·7 Å and a monoclinic form at 1·7 Å resolution, with crystallographic R-values of 0·199 and 0·192, respectively. The structure determination involved a novel application of restrained crystallographic refinement, in that the refinement of incomplete models was necessary in order to completely determine the course of the polypeptide chain. The recently determined amino acid sequence (Bloxham et al., 1981) has been fitted to the models. The molecule has substantially different conformations in the two crystal forms, and there is evidence that a conformational change is required for enzymatic activity.
A synthetic oligodeoxynucleotide probe was used to clone the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus. The sequence of a 2050-bp restriction fragment containing the gene was determined. Analysis of the gene-derived amino acid (aa) sequence showed that this exoenzyme is probably synthesized as a 283-aa precursor with a 24-aa signal peptide and a 14-aa propeptide. The mature, secreted enzyme comprises 245 aa residues. Sonicates of Escherichia coli HB101 carrying the gene on a multicopy plasmid showed phospholipase C activity. This activity was inhibited by Tris, a known inhibitor of the B. cereus enzyme and also by antiserum raised against pure B. cereus phospholipase C. We conclude therefore that the gene is expressed in E. coli. The cloning and sequencing described here complete the first step toward using in vitro mutagenesis for investigations of the structure-function relationships of B. cereus phospholipase C.
The previously published three-dimensional structure of porcine pancreatic prophospholipase A2 at 3resolution was found to be incompatible with the structures of bovine phospholipase A2 and bovine prophospholipase A2. This was unexpected because of the very homologous amino acid sequences of these enzymes. Therefore, the crystal structure of the porcine enzyme was redetermined using molecular replacement methods with bovine phospholipase as the parent model. The structure was crystallographically refined at 2·6resolution by fast Fourier transform and restrained least-squares procedures to an R-factor of 0·241.The crystals appeared to contain phospholipase A2 and not prophospholipase A2. Apparently the protein is slowly converted under the crystallization conditions employed. Our investigation shows that, in contrast to the previous report, the three-dimensional structure of porcine phospholipase A2 is very similar to that of bovine phospholipase A2, including the active site. Smaller differences were observed in some residues involved in the binding of aggregated substrates. However, an appreciable conformational difference is in the loop 59 to 70, where a single substitution at position 63 (bovine Val→porcine Phe) causes a complete rearrangement of the peptide chain.In addition to the calcium ion in the active site, a second calcium ion is present in the crystals; this is located on a crystallographic 2-fold axis and stabilizes the interaction between two neighbouring molecules.
Nous avons étudié des exo-enzymes de Bacillus Cereus, phospholipase-C et ß-lactamase II, par zinc et cobalt K-edge EXAFS. Nous rapportons que les zincs dans les centre structural et catalytique de phospholipase-C ont cinq ligants, tandis que le cobalt, substitué dans le centre catalytique, a six ligants. Le zinc dans le centre actif de ß-lactamase II a un ligant soufre, à bas nombre de coordination ou haut facteur Debye-Waller, outre des ligants imidazoles. We have studied the Bacillus Cereus exo-enzymes, phospholipase-C and ß-lactamase II, by Zn and Co K-edge EXAFS. We report that the zincs in the structural and catalytic sites of the former enzyme are 5-coordinate, whereas cobalt, substituted in the catalytic site, is 6 coordinate. The ß-lactamase II active site zinc has sulphur coordination, with either low occupancy or high Debye-Waller factor, in addition to imidazole coordination.
Crystals of phospholipase C from Bacillus cereus have been grown and are suitable for X-ray diffraction analysis to at least 2.2 Å. The crystals belong to the tetragonal space group P41 (or P43); unit cell dimensions are . The unit cell appears to contain 57% solvent and the asymmetric unit one molecule of enzyme.
1. When heated in 8 M-urea, phospholipase C(EC 3.1.4.3) from Bacillus cereus undergoes conformational transitions depending on the temperatures used. These transitions were studied by examining protein fluorescence, iodide quenching of protein fluorescence, u.v. difference spectroscopy, chemical availability of histidine residues in the enzyme, circular dichroism and catalytic activity. 2. Unless simultaneously exposed to elevated temperatures the enzyme appears to be unaffected by 8 M-urea. Removal of the two zinc atoms from the enzyme renders phospholipase C very sensitive to denaturation by 8 M-urea as indicated by fluorescence emission spectra and circular dichroism. 3. Both the native and the zinc-free enzymes are markedly more resistant to irreversible thermal inactivation in the presence of 8 M-urea than in its absence. 4. The response of the enzyme to 8 M-urea and the role of zinc in stabilizing the enzyme are discussed.
1. Protein-fluorescence studies indicated that phospholipase C from Bacillus cereus is denatured in solutions of guanidinium chloride. The denaturation was not thermodynamically reversible and followed biphasic kinetics. 2. Guanidinium chloride solutions released the structural Zn2+ from the enzyme and rendered all histidine residues chemically reactive. In the presence of free Zn1+ the enzyme was much more resistant to denaturation. Also, the addition for free Zn2+ to the denatured enzyme induced refolding. 3. The Zn2+-free apoenzyme was much more sensitive to guanidinium chloride than was the native enzyme and the denaturation appeared to be thermodynamically reversible. 4. Guanidinium chloride denaturation was associated with a reversible inactivation of the enzyme. Heat-inactivated, coagulated enzyme was substantially re-activated on dissolution in guanidinium chloride solutions followed by dialysis against a Zn2+-containing buffer.
1. The zinc content and metal ion dependence of phospholipase C(phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus have been examined. 2. The native enzyme contained about 2 atoms of tightly bound zinc/molecule. 3. Incubation of the enzyme with EDTA or with o-phenanthroline caused inactivation. The inactivation was accompanied by the removal of one zinc atom from the enzyme and could be fully reversed by the addition of Zn2+ or Co2+ to the enzyme and partly reversed by Mn2+ or Mg2+. 4. Prolonged exposure to o-phenanthroline removed the second zinc atom also and produced an enzyme species which was reactivated by Zn2+ only. Full reactivation was accompanied by the binding of about two zinc atoms to the enzyme. 5. The results are consistent with the view that phospholipase C is a zinc metalloenzyme.
A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.
The coordination sphere of both the structural and catalytic zinc ions of Bacillus cereus phospholipase C has been probed by substitution of cobalt(II) for zinc and investigation of the resultant derivatives by a variety of spectroscopic techniques. The electronic absorption, circular dichroic, magnetic circular dichroic, and electron paramagnetic resonance spectra were found to be strikingly similar when cobalt(II) was substituted into either site and are consistent with a distorted octahedral environment for the metal ion in both sites. Octahedral coordination appears comparatively rare in zinc metalloenzymes but has been suggested for glyoxalase I [Sellin, S., Eriksson, L. E. G., Aronsson, A.-C., & Mannervik, B. (1983) J. Biol. Chem. 258, 2091-2093; Garcia-Iniguez, L., Powers, L., Chance, B., Sellin, S., Mannervik, B., & Mildvan, A. S. (1984) Biochemistry 23, 685-689], transcarboxylase [Fung, C.-H., Mildvan, A. S., & Leigh, J. S. (1974) Biochemistry 13, 1160-1169], and the regulatory binding site of Aeromonas aminopeptidase [Prescott, J. M., Wagner, F. W., Holmquist, B., & Vallee, B. L. (1985) Biochemistry 24, 5350-5356]. Phospholipase C is so far unique in having two such sites.
Treatment of rat liver cells (the C-9 cell line), porcine aorta endothelial cells, bovine aorta smooth muscle cells, bovine aorta endothelial cells, mouse fibroblasts and rat keratinocytes with highly purified, crystallized Bacillus cereus phospholipase C, which hydrolyzes phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine but has little or no effect on phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, lysophosphatidylcholine or lysophosphatidylethanolamine, increased metabolism of arachidonic acid. Hydrolysis of phosphatidylcholine (and/or phosphatidylethanolamine) by a phosphatidylcholine (or phosphatidylethanolamine)-hydrolyzing phospholipase C appears to contribute to liberation of substrate for arachidonic acid metabolism.
The molecular model of myohemerythrin, an oxygen-carrying protein from sipunculan worms, has been refined by stereochemically restrained least-squares minimization at 1.7/1.3 A resolution to a conventional R-value of 0.158. The estimated positional standard deviation is better than 0.15 A for most of the 979 protein atoms. The average isotropic displacement parameter, B, for the protein atoms is 23.1 A2. This high average B parameter appears to be due to the overall motion of the molecule, which correlates with the observed anisotropic diffraction. The side-chains of seven residues were modeled in two conformations, i.e. the side-chains were discretely disordered, and B parameters for several lysine and glutamate side-chains indicate that they are poorly localized. Of the residues in myohemerythrin, 66% are helical, with 62% occurring in four long alpha-helices with mean values for the backbone torsion angles of phi = -65 degrees, psi = -42 degrees, and for the hydrogen bonds distances of N ... O, 3.0 A and H ... O, 2.1 A, and angles of N ... O = C, 153 degrees, N-H ... O, 157 degrees, and H ... O = C, 147 degrees. For two-thirds of the alpha-helical residues, the torsional rotation of the C alpha-C beta bond, chi 1, is approximately -60 degrees, and for one-third chi 1 is approximately 180 degrees. Although most turns in myohemerythrin are well-categorized by previous classification, two do not fit in established patterns. Also included in the refined model are three sulfate ions, all partially occupied, and 157 water molecules, 40% of which are modeled fully occupied. Only one water molecule is internal to the protein, the remainder occur on the surface and are observed principally between symmetry-related molecules contributing, along with van der Waals' contacts, most of the interactions between molecules. There are eight intermolecular protein-protein hydrogen bonds, of which only four are between well-located atoms.
The three-dimensional structure analysis of crystalline fungal catalase from Penicillium vitale has been extended to 2.0 A resolution. The crystals belong to space group P3(1)21, with the unit cell parameters of a = b = 144.4 A and c = 133.8 A. The asymmetric unit contains half a tetrameric molecule of 222 symmetry. Each subunit is a single polypeptide chain of approximately 670 amino acid residues and binds one heme group. The amino acid sequence has been tentatively determined by computer graphics model building (using the FRODO system) and comparison with the known sequence of beef liver catalase. The atomic model has been refined by the Hendrickson & Konnert (1981) restrained least-squares program against 68,000 reflections between 5 A and 2 A resolution. The final R-factor is 0.31 after 24 refinement cycles. The secondary and tertiary structure of the catalase has been analyzed.
Bovine pancreatic deoxyribonuclease I (DNase I), an endonuclease that degrades double-stranded DNA in a nonspecific but sequence-dependent manner, has been used as a biochemical tool in various reactions, in particular as a probe for the structure of chromatin and for the helical periodicity of DNA on the nucleosome and in solution. Limited digestion by DNase I, termed DNase I 'footprinting', is routinely used to detect protected regions in DNA-protein complexes. Recently, we have solved the three-dimensional structure of this glycoprotein (relative molecular mass 30,400) by X-ray structure analysis at 2.5 A resolution and have subsequently refined it crystallographically at 2.0 A. Based on the refined structure and the binding of Ca2+-thymidine 3',5'-diphosphate (Ca-pTp) at the active site, we propose a mechanism of action and present a model for the interaction of DNase I with double-stranded DNA that involves the binding of an exposed loop region in the minor groove of B-DNA and electrostatic interactions of phosphates from both strands with arginine and lysine residues on either side of this loop. We explain DNase I cleavage patterns in terms of this model and discuss the consequences of the extended DNase I-DNA contact region for the interpretation of DNase I footprinting results.
The chapter focuses on the practical application of stereochemically-restrained refinement to macromolecular crystals. Details of computational procedures and minimization algorithms are treated and need not be considered in routine applications. However, it is important to understand the nature of the function being minimized. Thorough structural refinement has become an integral part of macromolecular crystallography. The chapter describes some extensions of the current export versions of the programs that have been implemented or are envisioned. Atomic motion and conformational heterogeneity (or disorder) is major impediments to successful refinement. The use of Fourier transformations to compute structure factors and gradient vectors might greatly improve speed for large problems. There are numerous other improvements that can be envisioned including a method for modeling the fluid solvent, an appropriate treatment of the correlation between occupancy and thermal parameters of discrete solvent molecules, restraints for nonbonded contacts from crystal packing, inclusion of attractive potentials for nonbonded contacts, provision for refining partial structures, and proper estimation of standard deviations. Extensions such as these are expected to be important in realizing the goal of producing refined structural models that reproduce the diffraction patterns to within the accuracy of the measured data and which are compatible with prior stereochemical knowledge of macromolecules.
The 3.3-A resolution crystal structure of the large proteolytic fragment of Escherichia coli DNA polymerase I complexed with deoxythymidine monophosphate consists of two domains, the smaller of which binds zinc-deoxythymidine monophosphate. The most striking feature of the larger domain is a deep crevice of the appropriate size and shape for binding double-stranded B-DNA. A flexible subdomain may allow the enzyme to surround completely the DNA substrate, thereby allowing processive nucleotide polymerization without enzyme dissociation.
The structure of alkaline phosphatase from Escherichia coli has been determined to 2.8 Å resolution. The multiple isomorphous replacement electron density map of the dimer at 3.4 Å was substantially improved by molecular symmetry averaging and solvent flattening. From these maps, polypeptide chains of the dimer were built using the published amino acid sequence. Stereochemically restrained least-squares refinement of this model against native data, starting with 3.4 Å data and extending in steps to 2.8 Å resolution, proceeded to a final overall crystallographic R factor of 0.256.
A simplified method for the purification of phospholipase C from Bacillus cereus is described. The enzyme is homogeneous in disc electrophoresis and in dodecylsulphate-polyacrylamide gel electrophoresis as well as in the analytical ultracentrifuge. The enzyme had a molecular weight of 23000, pI= 6.5 and consisted of one subunit. A divalent metal ion was necessary, Zn2+ was the most active. The activation of factor VII by tissue thromboplastin was reversible when tissue thromboplastin was destroyed by phospholipase C.
The crystal structure of bovine carboxypeptidase A (Cox) has been refined at 1.54 A resolution using the restrained least-squares algorithm of Hendrickson & Konnert (1981). The crystallographic R factor (formula; see text) for structure factors calculated from the final model is 0.190. Bond lengths and bond angles in the carboxypeptidase A model have root-mean-square deviations from ideal values of 0.025 A and 3.6 degrees, respectively. Four examples of a reverse turn like structure (the "Asx" turn) requiring an aspartic acid or asparagine residue are observed in this structure. The Asx turn has the same number of atoms as a reverse turn, but only one peptide bond, and the hydrogen bond that closes the turn is between the Asx side-chain CO group and a main-chain NH group. The distributions of CO-N and NH-O hydrogen bond angles in the alpha-helices and beta-sheet structures of carboxypeptidase A are centered about 156 degrees. A total of 192 water molecules per molecule of enzyme are included in the final model. Unlike the hydrogen bonding geometry observed in the secondary structure of the enzyme, the CO-O(wat) hydrogen bond angle is distributed about 131 degrees, indicating the role of the lone pair electrons of the carbonyl oxygen in the hydrogen bond interaction. Twenty four solvent molecules are observed buried within the protein. Several of these waters are organized into hydrogen-bonded chains containing up to five waters. The average temperature factor for atoms in carboxypeptidase A is 8 A2, and varies from 5 A2 in the center of the protein, to over 30 A2 at the surface.
The structure of the thermostable protease thermolysin has been refined by a restrained least-squares procedure at a nominal resolution of 1.6 Å to a conventional R-value of 21.3% for 34,671 observed reflections (or R = 19.5% for reflections with F0 > 3σ(F0)). The refined structure was constrained to adhere to known stereochemistry, with root-mean-square deviations of 0.021 Å from ideal bond lengths and 2.9 ° from ideal bond angles. The final model included 173 solvent molecules, which were given unit occupancies. Seven of these are “buried” within the protein. Atoms with the least apparent thermal motion tend to be those that are most deeply buried within the two domains of the structure. The active-site zinc is shown to have approximately tetrahedral co-ordination. Unusual features of the structure, confirmed by the refinement, include a cis-proline, a γ-turn, and a single turn of left-handed α-helix. The refinement shows that thermolysin does not contain unusual structures and supports our previous assertion that the thermostability of thermolysin and of thermostable proteins in general is due to a combination of factors which, in different instances, can include hydrophobic interactions, hydrogen bonding, ionic interactions, disulfide linkages, metal binding and other forms of stabilization.
The three-dimensional structure of beef liver catalase has been determined to 2.5 å resolution by a combination of isomorphous and molecular replacement techniques. Heavy-atom positions were found using vector search and difference Fourier methods. The tetrameric catalase molecule has 222 symmetry with one of its dyads coincident with a crystallographic 2-fold axis. The known polypeptide sequence has been unambiguously fitted to the electron density map. The heme is well buried in a hydrophobic pocket, 20 Å below the surface of the molecule, and accessible through a hydrophobic channel. Residues that line the heme pocket belong to two different subunits. Tyr357 is the proximal heme ligand and the catalytically important residues on the distal side are residues His74 and Asnl47. The tertiary structure consists of four domains: an extended non-globular amino-terminal arm, which stabilizes the quaternary structure; an anti-parallel, eight-stranded β-barrel providing the residues on the distal side of the heme; a rather random “wrapping domain” around the subunit exterior including the proximal heme ligand; and a final λ-helical structure resembling the E, F, G and H helices of the globins.