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ABSTRACT: NADP(+) dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) belongs to a large family of α-hydroxyacid oxidative β-decarboxylases that catalyze similar three-step reactions, with dehydrogenation to an oxaloacid intermediate preceding β-decarboxylation to an enol intermediate followed by tautomerization to the final α-ketone product. A comprehensive view of the induced fit needed for catalysis is revealed on comparing the first "fully closed" crystal structures of a pseudo-Michaelis complex of wild-type Escherichia coli IDH (EcoIDH) and the "fully closed" reaction product complex of the K100M mutant with previously obtained "quasi-closed" and "open" conformations. Conserved catalytic residues, binding the nicotinamide ring of NADP(+) and the metal-bound substrate, move as rigid bodies during domain closure by a hinge motion that spans the central β-sheet in each monomer. Interactions established between Thr105 and Ser113, which flank the "phosphorylation loop", and the nicotinamide mononucleotide moiety of NADP(+) establish productive coenzyme binding. Electrostatic interactions of a Lys100-Leu103-Asn115-Glu336 tetrad play a pivotal role in assembling a catalytically competent active site. As predicted, Lys230* is positioned to deprotonate/reprotonate the α-hydroxyl in both reaction steps and Tyr160 moves into position to protonate C3 following β-decarboxylation. A proton relay from the catalytic triad Tyr160-Asp307-Lys230* connects the α-hydroxyl of isocitrate to the bulk solvent to complete the picture of the catalytic mechanism.
Biochemistry 08/2012; 51(36):7098-115. · 3.42 Impact Factor
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ABSTRACT: RuvBL1 (RuvB-like 1) and its homolog RuvBL2 are evolutionarily highly conserved AAA(+) ATPases essential for many cellular activities. They play an important role in chromatin remodeling, transcriptional regulation and DNA damage repair. RuvBL1 and RuvBL2 are overexpressed in different types of cancer and interact with major oncogenic factors, such as β-catenin and c-Myc regulating their function. We solved the first three-dimensional crystal structure of the human RuvBL complex with a truncated domain II and show that this complex is competent for helicase activity. The structure reveals a dodecamer consisting of two heterohexameric rings with alternating RuvBL1 and RuvBL2 monomers bound to ADP/ATP, that interact with each other via the retained part of domain II. The dodecameric quaternary structure of the R1ΔDII/R2ΔDII complex observed in the crystal structure was confirmed by small-angle X-ray scattering analysis. Interestingly, truncation of domain II led to a substantial increase in ATP consumption of RuvBL1, RuvBL2 and their complex. In addition, we present evidence that DNA unwinding of the human RuvBL proteins can be auto-inhibited by domain II, which is not present in the homologous bacterial helicase RuvB. Our data give new insights into the molecular arrangement of RuvBL1 and RuvBL2 and strongly suggest that in vivo activities of these highly interesting therapeutic drug targets are regulated by cofactors inducing conformational changes via domain II in order to modulate the enzyme complex into its active state.
Journal of Structural Biology 09/2011; 176(3):279-91. · 3.41 Impact Factor
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ABSTRACT: Klebsiella pneumoniae is a nosocomial pathogen frequently isolated from opportunistic infections, especially in clinical environments. In spite of its potential pathogenicity, this microorganism has several metabolic potentials that could be used in biotechnology applications. K. pneumoniae is able to metabolize glycerol as a sole source of carbon and energy. 1,3-Propanediol dehydrogenase is the core of the metabolic pathway for the use of glycerol. We have determined the crystallographic structure of 1,3-propanediol dehydrogenase, a type III Fe-NAD-dependent alcohol dehydrogenase, at 2.7-A resolution. The structure of the enzyme monomer is closely related to that of other alcohol dehydrogenases. The overall arrangement of the enzyme showed a decameric structure, formed by a pentamer of dimers, which is the catalytic form of the enzyme. Dimers are associated by strong ionic interactions that are responsible for the highly stable in vivo packing of the enzyme. Kinetic properties of the enzyme as determined in the article would suggest that this decameric arrangement is related to the cooperativity between monomers.
Journal of bacteriology 12/2008; 191(4):1143-51. · 3.94 Impact Factor
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ABSTRACT: The complex of RuvBL1 and its homologue RuvBL2, two evolutionarily highly conserved eukaryotic proteins belonging to the AAA(+) (ATPase associated with diverse cellular activities) family of ATPases, was co-expressed in Escherichia coli. For crystallization purposes, the flexible domains II of RuvBL1 and RuvBL2 were truncated. The truncated RuvBL1-RuvBL2 complex was crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystals were hexagonal-shaped plates and belonged to either the orthorhombic space group C222(1), with unit-cell parameters a = 111.4, b = 188.0, c = 243.4 A and six monomers in the asymmetric unit, or the monoclinic space group P2(1), with unit-cell parameters a = 109.2, b = 243.4, c = 109.3 A, beta = 118.7 degrees and 12 monomers in the asymmetric unit. The crystal structure could be solved by molecular replacement in both possible space groups and the solutions obtained showed that the complex forms a dodecamer.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2008; 64(Pt 9):840-6. · 0.51 Impact Factor
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ABSTRACT: Flavodiiron proteins (FDP) are modular enzymes which function as NO and/or O(2) reductases. Although the majority is composed of two structural domains, the homolog found in Escherichia coli, flavorubredoxin, possesses an extra C-terminal module consisting of a linker and a rubredoxin (Rd) domain necessary for interprotein redox processes. In order to investigate the location of the Rd domain with respect to the flavodiiron structural core, small-angle X-ray scattering was used to construct low-resolution structural models of flavorubredoxin. Scattering patterns from the Rd domain, the FDP core, and full-length flavorubredoxin were collected. The latter two species were found to be tetrameric in solution. Ab initio shape reconstruction and rigid-body modeling indicate a peripheral location for the Rd domains, which appear to have weak contacts with the FDP core. This finding suggests that Rd behaves as an independent domain and is freely available to participate in redox reactions with protein partners.
Structure 10/2008; 16(9):1428-36. · 6.35 Impact Factor
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ABSTRACT: Two Bacillus subtilis extracellular endo-1,5-alpha-L-arabinanases, AbnA and Abn2, belonging to glycoside hydrolase family 43 have been identified. The recently characterized Abn2 protein hydrolyzes arabinan and has low identity to other reported 1,5-alpha-L-arabinanases. Abn2 and its selenomethionine (SeMet) derivative have been purified and crystallized. Crystals appeared in two different space groups: P1, with unit-cell parameters a = 51.9, b = 57.6, c = 86.2 A, alpha = 82.3, beta = 87.9, gamma = 63.6 degrees , and P2(1)2(1)2(1), with unit-cell parameters a = 57.9, b = 163.3, c = 202.0 A. X-ray data have been collected for the native and the SeMet derivative to 1.9 and 2.7 A resolution, respectively. An initial model of Abn2 is being built in the SeMet-phased map.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2008; 64(Pt 7):636-8. · 0.51 Impact Factor
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ABSTRACT: The hybrid cluster protein (HCP) from the sulfate-reducing bacterium Desulfovibrio vulgaris strain Hildenborough has been isolated and crystallized anaerobically. The phase problem was solved for a P2(1)2(1)2(1) crystal form using multiple-wavelength anomalous diffraction data collected in the vicinity of the Fe K absorption edge. Although the overall protein structure is essentially the same as that previously obtained, it shows that the nature of the hybrid cluster has particular differences when isolated and crystallized in the absence of oxygen and this provides insight into the structural features associated with changes in the oxidation state. A comparison between HCPs and carbon monoxide dehydrogenases (CoDs) shows that they possess a similar fold and that the dehydrogenases have a related cluster at the equivalent HCP hybrid cluster position. This helps to understand the nature of the hybrid cluster and to predict a dimeric structure for class 3 HCPs, which lack the N-terminal region.
Acta Crystallographica Section D Biological Crystallography 07/2008; 64(Pt 6):665-74. · 12.62 Impact Factor
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ABSTRACT: Spores of Bacillus subtilis are covered by a multi-protein protective coat which is a key factor in their extreme environmental resilience. A fraction of the coat proteins undergoes covalent cross-linking following their assembly at the spore surface. Several types of covalent cross-links are found in the coat. These include epsilon-(gamma-glutamyl)lysine bonds whose formation is catalyzed by a transglutaminase, Tgl, itself a coat component. Tgl is the smallest known transglutaminase. It bears no sequence resemblance to other proteins in databases, except for its counterparts in other Bacillus and related species, suggesting a highly specialized role in coat assembly. It is not known to what degree are the Tgl-like proteins structural and mechanistically related to other transglutaminases. Here, we have fused the His(6) tag to the C-terminal end of Tgl, and shown that the fusion protein is functional in vivo. We have overproduced B. subtilis Tgl-His(6) by auto-induction with high yield and purified the protein to nearly homogeneity in a single chromatographic step. The purified protein, active as it catalyzed the cross-linking of bovine serum albumin, behaved as a monomer of about 33kDa in solution. Lastly, Tgl was crystallized and X-ray diffraction data were collected using synchrotron radiation to 2.1A resolution. Crystals of Tgl belong to the tetragonal space group P4(1,3) and contain two molecules per asymmetric unit.
Protein Expression and Purification 06/2008; 59(1):1-8. · 1.59 Impact Factor
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ABSTRACT: Detailed structural models of di-cluster seven-iron ferredoxins constitute a valuable resource for folding and stability studies relating the metal cofactors' role in protein stability. The here reported, hemihedric twinned crystal structure at 2.0 A resolution from Acidianus ambivalens ferredoxin, shows an integral 103 residues, physiologically relevant native form composed by a N-terminal extension comprising a His/Asp Zn(2+) site and the ferredoxin (betaalphabeta)(2) core, which harbours intact clusters I and II, a [3Fe-4S](1+/0) and a [4Fe-4S](2+/1+) centres. This is in contrast with the previously available ferredoxin structure from Sulfolofus tokodai, which was obtained from an artificial oxidative conversion with two [3Fe-4S](1+/0) centres and poor definition around cluster II.
FEBS Letters 04/2008; 582(5):763-7. · 3.54 Impact Factor
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ABSTRACT: Crystallographic studies on flavodiiron proteins (FDPs) have revealed that the common sequence core ( approximately 400 residues) that defines this protein family comprises two structural domains. The N-terminal domain (of approximately 250 residues) displays a metallo-beta-lactamase-like-fold, being indeed structurally homologous to beta-lactamases and glyoxalases, despite the poor sequence similarity. Whereas beta-lactamases have mono- or dizinc sites and glyoxalases a mixed iron-zinc site, the lactamase domain of FDPs harbors a nonheme diiron center with carboxylate and histidine residues as ligands, assigned as the active site of NO and/or O(2) reduction. The C-terminal domain of FDPs is characterized by a flavodoxin-like fold, homologous to short-chain flavodoxins, and harbors a flavin mononucleotide moiety, stabilized by van der Waals interactions and a number of hydrogen bonds. Structures of FDPs obtained in different conditions and oxidation states display some heterogeneities, mostly at the diiron site, but still fail to provide unequivocal evidence for some pending questions regarding the substrate activation mechanism of FDPs, namely the preference for either substrate (NO or oxygen) observed in different members of this protein family. More structural studies are therefore required to achieve a deeper understanding on these matters.
Methods in Enzymology 02/2008; 437:3-19. · 2.04 Impact Factor
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12/2007; , ISBN: 9780470028636
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ABSTRACT: RuvBL1 is an evolutionarily highly conserved eukaryotic protein belonging to the AAA(+)-family of ATPases (ATPase associated with diverse cellular activities). It plays important roles in essential signaling pathways such as the c-Myc and Wnt pathways in chromatin remodeling, transcriptional and developmental regulation, and DNA repair and apoptosis. Herein we present the three-dimensional structure of the selenomethionine variant of human RuvBL1 refined using diffraction data to 2.2A of resolution. The crystal structure of the hexamer is formed of ADP-bound RuvBL1 monomers. The monomers contain three domains, of which the first and the third are involved in ATP binding and hydrolysis. Although it has been shown that ATPase activity of RuvBL1 is needed for several in vivo functions, we could only detect a marginal activity with the purified protein. Structural homology and DNA binding studies demonstrate that the second domain, which is unique among AAA(+) proteins and not present in the bacterial homolog RuvB, is a novel DNA/RNA-binding domain. We were able to demonstrate that RuvBL1 interacted with single-stranded DNA/RNA and double-stranded DNA. The structure of the RuvBL1.ADP complex, combined with our biochemical results, suggest that although RuvBL1 has all the structural characteristics of a molecular motor, even of an ATP-driven helicase, one or more as yet undetermined cofactors are needed for its enzymatic activity.
Journal of Biological Chemistry 01/2007; 281(50):38918-29. · 4.77 Impact Factor
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04/2006; , ISBN: 9780470028636
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ABSTRACT: The crystal structures of the oxidized and reduced forms of cytochrome c" from Methylophilus methylotrophus were solved from X-ray synchrotron data to atomic resolution. The overall fold of the molecule in the two redox states is very similar and is comparable to that of the oxygen-binding protein from the purple phototrophic bacterium Rhodobacter sphaeroides. However, significant modifications occur near the haem group, in particular the detachment from axial binding of His95 observed upon reduction as well as the adoption of different conformations of some protonatable residues that form a possible proton path from the haem pocket to the protein surface. These changes are associated with the previously well characterized redox-Bohr behaviour of this protein. Furthermore they provide a model for one of the presently proposed mechanisms of proton translocation in the much more complex protein cytochrome c oxidase.
JBIC Journal of Biological Inorganic Chemistry 04/2006; 11(2):189-96. · 3.29 Impact Factor
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ABSTRACT: RuvBL1, an evolutionary highly conserved protein related to the AAA+ family of ATPases, has been crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystals are hexagonal and belong to space group P6, with unit-cell parameters a = b = 207.1, c = 60.7 A and three molecules in the asymmetric unit.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2006; 62(Pt 1):61-6. · 0.51 Impact Factor
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Maria Luisa Rodrigues,
Margarida Archer,
Paulo Martel,
Sandra Miranda,
Mónica Thomaz,
Francisco Javier Enguita,
Ricardo P Baptista,
Eduardo Pinho e Melo,
Nelson Sousa,
Alfredo Cravador, Maria Arménia Carrondo
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ABSTRACT: The crystal structure of the elicitin beta-cinnamomin (beta-CIN) was determined in complex with ergosterol at 1.1 A resolution. beta-CIN/ergosterol complex crystallized in the monoclinic space group P2(1), with unit cell parameters of a = 31.0, b = 62.8, c = 50.0 A and beta = 93.4 degrees and two molecules in the asymmetric unit. Ligand extraction with chloroform followed by crystallographic analysis yielded a 1.35 A structure of beta-CIN (P4(3)2(1)2 space group) where the characteristic elicitin fold was kept. After incubation with cholesterol, a new complex structure was obtained, showing that the protein retains, after the extraction procedure, its ability to complex sterols. The necrotic effect of beta-CIN on tobacco was also shown to remain unchanged. Theoretical docking studies of the triterpene lupeol to beta-CIN provided an explanation for the apparent inability of beta-CIN to bind this ligand, as observed experimentally.
Biochimica et Biophysica Acta 02/2006; 1764(1):110-21. · 4.66 Impact Factor
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ABSTRACT: The multi-copper oxidases oxidise substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. The precise mechanism of this reduction has been unclear, but recent X-ray structural studies using the CotA endospore coat protein from Bacillus subtilis have given further insights into the principal stages. It is proposed that the mechanism involves binding of the dioxygen into the trinuclear centre so that it is sited approximately symmetrically between the two type 3 copper ions with one oxygen atom close to the type 2 copper ion. Further stages involve the formation of a peroxide intermediate and following the splitting of this intermediate, the migration of the hydroxide moieties towards the solvent exit channel. The migration steps are likely to involve a movement of the type 2 copper ion and its environment. Details of a putative mechanism are described herein based both on structures already reported in the literature and on structures of the CotA protein in the oxidised and reduced states and with the addition of peroxide and the inhibitor, azide.
Dalton Transactions 12/2005; · 3.84 Impact Factor
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ABSTRACT: Sulphate-reducing organisms are widespread in anaerobic enviroments, including the gastrointestinal tract of man and other animals. The study of these bacteria has attracted much attention over the years, due also to the fact that they can have important implications in industry (in biocorrosion and souring of oil and gas deposits), health (in inflamatory bowel diseases) and the environment (bioremediation). The characterization of the various components of the electron transport chain associated with the hydrogen metabolism in Desulfovibrio has generated a large and comprehensive list of studies. This review summarizes the more relevant aspects of the current information available on the structural data of various molecules associated with hydrogen metabolism, namely hydrogenases and cytochromes. The transmembrane redox complexes known to date are also described and discussed. Redox-Bohr and cooperativity effects, observed in a few cytochromes, and believed to be important for their functional role, are discussed. Kinetic studies performed with these redox proteins, showing clues to their functional inter-relationship, are also addressed. These provide the groundwork for the application of a variety of molecular modelling approaches to understanding electron transfer and protein interactions among redox partners, leading to the characterization of several transient periplasmic complexes. In contrast to the detailed understanding of the periplasmic hydrogen oxidation process, very little is known about the cytoplasmic side of the respiratory electron transfer chain, in terms of molecular components (with exception of the terminal reductases), their structure and the protein-protein interactions involved in sulphate reduction. Therefore, a thorough understanding of the sulphate respiratory chain in Desulfovibrio remains a challenging task.
Progress in Biophysics and Molecular Biology 12/2005; 89(3):292-329. · 3.20 Impact Factor
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ABSTRACT: Crystals of the title protein have been produced and preliminary structural analysis has been carried out. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 258.1, b = 340.1, c = 266.5 A. The protein forms a 24-mer of 20 kDa subunits, which assemble with 432 non-crystallographic symmetry. A total of 36 monomers are found in the asymmetric unit, corresponding to one and a half 24-mers.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2005; 61(Pt 5):503-6. · 0.51 Impact Factor
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Ilídio J Correia,
Catarina M Paquete,
Ana Coelho,
Claudia C Almeida,
Teresa Catarino,
Ricardo O Louro,
Carlos Frazão,
Lígia M Saraiva, Maria Arménia Carrondo,
David L Turner,
António V Xavier
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ABSTRACT: The tetraheme cytochrome c3 isolated from Desulfomicrobium baculatum (DSM 1743)(Dsmb) was cloned, and the sequence analysis showed that this cytochrome differs in just three amino acid residues from the cytochrome c3 isolated from Desulfomicrobium norvegicum (Dsmn): (DsmnXXDsmb) Thr-37 --> Ser, Val-45 --> Ala, and Phe-88 --> Tyr. X-ray crystallography was used to determine the structure of cytochrome c3 from Dsmb, showing that it is very similar to the published structure of cytochrome c3 from Dsmn. A detailed thermodynamic and kinetic characterization of these two tetraheme cytochromes c3 was performed by using NMR and visible spectroscopy. The results obtained show that the network of cooperativities between the redox and protonic centers is consistent with a synergetic process to stimulate the hydrogen uptake activity of hydrogenase. This is achieved by increasing the affinity of the cytochrome for protons through binding electrons and, reciprocally, by favoring a concerted two-electron transfer assisted by the binding of proton(s). The data were analyzed within the framework of the differences in the primary and tertiary structures of the two proteins, showing that residue 88, close to heme I, is the main cause for the differences in the microscopic thermodynamic parameters obtained for these two cytochromes c3. This comparison reveals how replacement of a single amino acid can tune the functional properties of energy-transducing proteins, so that they can be optimized to suit the bioenergetic constraints of specific habitats.
Journal of Biological Chemistry 12/2004; 279(50):52227-37. · 4.77 Impact Factor
