Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.12.7.2 (hydrogenase)
3,522 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 microM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 microM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism.
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PMID:Zn2+-linked dimerization of UreG from Helicobacter pylori, a chaperone involved in nickel trafficking and urease activation. 1876 50

The high-affinity nickel-binding site of the Escherichia coli [NiFe]-hydrogenase accessory protein HypB was localized to residues at the immediate N-terminus of the protein. Modification of a metal-binding fusion protein, site-directed mutagenesis experiments, and DFT calculations were used to identify the N-terminal amine as a ligand as well as the three cysteine residues in the CXXCGCXXX motif. This sequence can be removed from the protein and both a synthesized peptide and a protein fusion bind nickel with a similar affinity and the same structure as the parent metalloprotein, indicating the self-sufficiency of this high-affinity nickel-binding sequence.
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PMID:A high-affinity metal-binding peptide from Escherichia coli HypB. 1883 29

The mechanism for H(2) cleavage in NiFe-hydrogenase has been reinvestigated with large models using both hybrid DFT by itself, or in a QM/MM scheme following the ONIOM approach. Heterolytic cleavage, with one hydrogen ending up as a bridging hydride and one as a proton on a cysteine ligand, was found to have a barrier slightly too high to be compatible with measured catalytic turnover rates. Alternative mechanisms were therefore investigated. In the finally suggested mechanism, heterolytic cleavage is used only as an initial step to generate a complex with nickel in oxidation state Ni(I). In the following cycles, H(2) is instead cleaved on nickel using an oxidative addition mechanism with a lower barrier. It was found that the ONIOM results for the reaction mechanism in NiFe-hydrogenase needed to be corrected by large model DFT results to be more reliable. This was mainly an effect of overestimation of polarization effects of the QM region by the MM region due to the particular treatment of the electrostatic interactions and the use of a standard (nonpolarizable) force field.
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PMID:An autocatalytic mechanism for NiFe-hydrogenase: reduction to Ni(I) followed by oxidative addition. 1913 2

Iron-sulfur (Fe/S) protein maturation in the eukaryotic cytosol and nucleus requires conserved components of the essential CIA machinery. The CIA protein Nar1 performs a specific function in transferring an Fe/S cluster that is assembled de novo on the Cfd1-Nbp35 scaffold to apoproteins. Here, we used systematic site-directed mutagenesis and a combination of in vitro and in vivo studies to show that Nar1 holds two Fe/S clusters at conserved N- and C-terminal cysteine motifs. A wealth of biochemical studies suggests that the assembly of these Fe/S clusters on Nar1 cannot be studied in Escherichia coli, as the recombinant protein does not contain the native Fe/S clusters. We therefore followed Fe/S cluster incorporation directly in yeast by a (55)Fe radiolabeling method in vivo, and we measured the functional consequences of Nar1 mutations in the assembly of cytosolic Fe/S proteins. We find that both Fe/S clusters are essential for Nar1 function and cell viability. Molecular modeling using a structurally but not functionally related bacterial iron-only hydrogenase as a template provided compelling structural explanations for our mutational data. The C-terminal Fe/S cluster is stably buried within Nar1, whereas the N-terminal one is exposed at the protein surface and hence may be more easily lost. Insertion of an Fe/S cluster into the C-terminal location depends on the N-terminal motif, suggesting the participation of the latter motif in the assembly process of the C-terminal cluster. The vicinity of the two Fe/S centers suggests a close functional cooperation during cytosolic Fe/S protein maturation.
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PMID:Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1. 1938 3

Entamoeba histolytica is known for its extraordinary capacity to destroy human tissues, leading to invasive diseases such as ulcerative colitis or extra-intestinal abscesses. In order to identify the virulence factors of this parasite phenotypes and proteomes of two recently identified genetically related cell lines (A and B), derived from the laboratory E. histolytica isolate HM-1:IMSS, were compared. Both cell lines are indistinguishable on the basis of highly polymorphic tandem repeat DNA sequences. However, cell line A is incapable to induce liver abscesses in experimentally infected rodents, whereas cell line B provokes considerable abscesses. Phenotypic analyses revealed increased hemolytic activity, lower growth rate, smaller cell size, reduced cysteine peptidase activity and lower resistance to nitric oxide stress for cell line A. In contrast, no differences between the two cell lines were found for cytopathic activity, erythrophagocytosis, digestion of erythrocytes or resistance to complement, hydrogen peroxide and superoxide radical anions. Proteomic comparison by 2-D DIGE followed by MS, identified a total of 21 proteins with higher abundance in cell line A and ten proteins with higher abundance in cell line B. Remarkably, three differentially up-regulated antioxidants were exclusively found in the pathogenic cell line B. Notably, only for two differentially regulated proteins, namely a Fe-hydrogenase and a C2 domain protein, a similar type was found at the level of transcription. Summarized, a defined set of different proteins could be identified between cell lines A and B. These molecules may have an important role in amoeba pathogenicity.
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PMID:Comparison of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties. 1968 50

A common approach for the computational modeling of enzyme reactions is to study a rather small model of the active site (20-200 atoms) with quantum mechanical (QM) methods, modeling the rest of the surroundings by a featureless continuum with a dielectric constant of approximately 4. In this paper, we discuss how the residues included in the QM model should be selected and how many residues need to be included before reaction energies converge. As a test case, we use a proton-transfer reaction between a first-sphere cysteine ligand and a second-sphere histidine group in the active site of [Ni,Fe] hydrogenase. We show that it is not a good approach to add groups according to their distance to the active site. A better approach is to add groups according to their contributions to the QM/MM energy difference. However, the energies can still vary by up to 50 kJ/mol for QM systems of sizes up to 230 atoms. In fact, the QM-only approach is based on the hope that a large number of sizable contributions will cancel. Interactions with neutral groups are, in general, short-ranged, with net energy contributions of less than 4 kJ/mol at distances above 5 A from the active site. Interactions with charged groups are much more long-ranged, and interactions with buried charges 20 A from the active site can still contribute by 5 kJ/mol to the reaction energy. Thus, to accurately model the influence of the surroundings on enzyme reaction energies, a detailed and unbiased atomistic account of the surroundings needs to be included.
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PMID:Do quantum mechanical energies calculated for small models of protein-active sites converge? 1978 74

Metallochaperones are essential for the safe and targeted delivery of necessary yet toxic metal cofactors to their respective protein partners. In this study we examine the nickel-binding properties of the Escherichia coli protein SlyD, a factor that contributes to optimal nickel accumulation in this organism. This protein is also required for E. coli energy metabolism because it participates in the nickel insertion step during [Ni-Fe]-hydrogenase metallocenter assembly. Our study demonstrates that SlyD is a multiple nickel ion binding protein. The analysis of noncovalent metal-protein complexes via electrospray ionization mass spectrometry revealed that SlyD binds up to seven nickel ions in a noncooperative manner with submicromolar affinity (<2 microM, upper limit) and that the protein exists in a dynamic mixture of metalloforms that is dependent on the availability of nickel ions in solution. Structural analysis indicates that this metallochaperone undergoes small but distinct changes in the structure upon metal binding and that the nickel-binding sites are assembled through beta-turn formation. Although the C-terminal metal-binding domain is primarily responsible for metal chelation, we find that metal binding also perturbs the structure of the N-terminal domains. An investigation of the nickel sites by using X-ray absorption spectroscopy shows that SlyD binds nickel ions by adapting several different geometries and coordination numbers. Finally, the characterization of SlyD mutants demonstrates that the cysteine residues in the C-terminal domain confer tighter affinity as well as increased binding capacity to SlyD. On the basis of the presented data a model for nickel binding to SlyD as well as its role in nickel homeostasis is discussed.
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PMID:The Ni(II)-binding properties of the metallochaperone SlyD. 1994 32

The generation of H(2) by the use of solar energy is a promising way to supply humankind's energy needs while simultaneously mitigating environmental concerns that arise due to climate change. The challenge is to find a way to connect a photochemical module that harnesses the sun's energy to a catalytic module that generates H(2) with high quantum yields and rates. In this review, we describe a technology that employs a "molecular wire" to connect a terminal [4Fe-4S] cluster of Photosystem I directly to a catalyst, which can be either a Pt nanoparticle or the distal [4Fe-4S] cluster of an [FeFe]- or [NiFe]-hydrogenase enzyme. The keys to connecting these two moieties are surface-located cysteine residues, which serve as ligands to Fe-S clusters and which can be changed through site-specific mutagenesis to glycine residues, and the use of a molecular wire terminated in sulfhydryl groups to connect the two modules. The sulfhydryl groups at the end of the molecular wire form a direct chemical linkage to a suitable catalyst or can chemically rescue a [4Fe-4S] cluster, thereby generating a strong coordination bond. Specifically, the molecular wire can connect the F(B) iron-sulfur cluster of Photosystem I either to a Pt nanoparticle or, by using the same type of genetic modification, to the differentiated iron atom of the distal [4Fe-4S].(Cys)(3)(Gly) cluster of hydrogenase. When electrons are supplied by a sacrificial donor, this technology forms the cathode of a photochemical half-cell that evolves H(2) when illuminated. If such a device were connected to the anode of a photochemical half-cell that oxidizes water, an in vitro solar energy converter could be realized that generates only O(2) and H(2) in the light. A similar methodology can be used to connect Photosystem I to other redox proteins that have surface-located [4Fe-4S] clusters. The controlled light-driven production of strong reductants by such systems can be used to produce other biofuels or to provide mechanistic insights into enzymes catalyzing multielectron, proton-coupled reactions.
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PMID:Wiring photosystem I for direct solar hydrogen production. 1994 49

The geometric and electronic structures of the active sites in the oxidized Ni(r)-B state of the [NiFe] hydrogenases from Ralstonia eutropha H16 and Desulfovibrio vulgaris Miyazaki F were investigated in pulsed EPR and ENDOR experiments at two different microwave frequencies (X- and Q-band). Two hyperfine-couplings were clearly resolved in the frozen solution spectra arising from the beta-protons of the nickel-coordinating cysteine residues Cys549 and Cys586 from the Desulfovibrio vulgaris and Ralstonia eutropha hydrogenase, respectively. ESEEM spectroscopic experiments reveal the presence of a histidine in the second coordination sphere of the Ni. The spectroscopic data indicate that the electronic structures of the [NiFe] centers in both hydrogenases are identical in the Ni(r)-B state. However, additional spin couplings of the active site to further paramagnetic centers were identified for the Ralstonia eutropha hydrogenase. The respective couplings could be clearly resolved and simulated. The results from this study are discussed in view of the exceptional O(2)-tolerance of the Ralstonia hydrogenase.
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PMID:Comparison of the membrane-bound [NiFe] hydrogenases from R. eutropha H16 and D. vulgaris Miyazaki F in the oxidized ready state by pulsed EPR. 2016 62

Complex enzymes containing Fe-S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe-4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non-protein ligands. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear. Here we show the structure of HydA(DeltaEFG) (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) revealing the presence of a [4Fe-4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe-4S] subcluster by generalized host-cell machinery and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes.
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PMID:Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA(DeltaEFG). 2041 61


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