Gene sequence and crystal structure of the aldehyde oxidoreductase from Desulfovibrio desulfuricans ATCC 27774 (original) (raw)
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Journal of Molecular Biology, 2000
The aldehyde oxidoreductase (MOD) isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) is a member of the xanthine oxidase family of molybdenum-containing enzymes. It has substrate speci-®city similar to that of the homologous enzyme from Desulfovibrio gigas (MOP) and the primary sequences from both enzymes show 68 % identity. The enzyme was crystallized in space group P6 1 22, with unit cell dimensions of a b 156.4 A Ê and c 177.1 A Ê , and diffraction data were obtained to beyond 2.8 A Ê . The crystal structure was solved by Patterson search techniques using the coordinates of the D. gigas enzyme. The overall fold of the D. desulfuricans enzyme is very similar to MOP and the few differences are mapped to exposed regions of the molecule. This is re¯ected in the electrostatic potential surfaces of both homologous enzymes, one exception being the surface potential in a region identi®able as the putative docking site of the physiological electron acceptor. Other essential features of the MOP structure, such as residues of the active-site cavity, are basically conserved in MOD. Two mutations are located in the pocket bearing a chain of catalytically relevant water molecules.
Journal of Molecular Biology, 2000
The aldehyde oxidoreductase (MOD) isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) is a member of the xanthine oxidase family of molybdenum-containing enzymes. It has substrate speci-®city similar to that of the homologous enzyme from Desulfovibrio gigas (MOP) and the primary sequences from both enzymes show 68 % identity. The enzyme was crystallized in space group P6 1 22, with unit cell dimensions of a b 156.4 A Ê and c 177.1 A Ê , and diffraction data were obtained to beyond 2.8 A Ê . The crystal structure was solved by Patterson search techniques using the coordinates of the D. gigas enzyme. The overall fold of the D. desulfuricans enzyme is very similar to MOP and the few differences are mapped to exposed regions of the molecule. This is re¯ected in the electrostatic potential surfaces of both homologous enzymes, one exception being the surface potential in a region identi®able as the putative docking site of the physiological electron acceptor. Other essential features of the MOP structure, such as residues of the active-site cavity, are basically conserved in MOD. Two mutations are located in the pocket bearing a chain of catalytically relevant water molecules.
Journal of the …, 2009
Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerolinhibited enzyme, where the catalytically labile OH/OH 2 ligand is lost and both alcohols coordinate the Mo site in a η 2 fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.
PLoS ONE, 2013
Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a g 2 fashion inhibiting the enzyme activity.
Thesis, North Carolina State University, 2018
CAREY, LEIAH MARIE. Probing the Structure-Function Relationship of a Multifunctional Enzyme using Crystallographic Diffraction Methods. (Under the direction of Dr. Reza Ghiladi.) The marine annelid Amphitrite ornata possesses the ability to chemically detoxify deleterious aromatic compounds prevalent in its environment, which is afforded by its coelomic hemoglobin, Dehaloperoxidase (DHP). In addition to its oxygen transport function, DHP also possesses peroxidase, peroxygenase, oxidase and oxygenase enzymatic functions, resulting in 5 activities that coexist at a single heme reactive center. Structurally, DHP possesses the canonical -helical globin fold associated with oxygen transport, yet lacks the structural homology with archetypical monofunctional examples of which it shares enzymatic reactivity, such as horseradish peroxidase, AaeAPO, cytochrome c oxidase and cytochrome
Critical Residues for Structure and Catalysis in Short-chain Dehydrogenases/Reductases
Journal of Biological Chemistry, 2002
Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10 -30%), the three-dimensional structures display a highly similar ␣/ folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3/17-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wildtype enzyme at 1.2-Å resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.
Toward a Structural Understanding of the Dehydratase Mechanism
Structure, 2002
making newly approved drugs useless within only a few years. There is thus a permanent need for the development of novel antibiotics, either through the variation of known principles or through targeting pathways so far unexploited. Many of the available antibiotics interfere with bacterial cell wall biosynthesis. The bacterial cell wall is not St. Andrews Scotland KY16 9ST only fundamentally different from its eukaryotic counterpart but also has a vital shielding function, and its com-United Kingdom 2 Joint Structural Biology Group ponents are often important virulence factors. Among the main components of the bacterial cell wall are com-ESRF F38043 Grenoble Cedex plex carbohydrate structures. One of the building blocks in a wide variety of both Gram-positive and Gram-nega-France 3 Department of Microbiology tive species is L-rhamnose, a 6-deoxyhexose that is not present in mammals. L-rhamnose has been found to University of Guelph Ontario N1G 2W1 occupy important anchoring positions, e.g., in Mycobacterium tuberculosis, where it covalently links the arabi-Canada 4 Zentrum fü r Ultrastrukturforschung und nogalactan to the peptidoglycan layer [1]. It has been demonstrated that loss of arabinogalactan leads to loss Ludwig Boltzmann-Institut fü r Molekulare Nanotechnologie of viability of this important pathogen [2, 3]. In a recent study, inhibitors of L-rhamnose-synthesizing enzymes Universitä t fü r Bodenkultur Wien A-1180 Vienna were shown to possess activity against whole M. tuberculosis cells [4]. Gram-negative bacteria utilize L-rham-Austria nose in their lipopolysaccharide (LPS). For example, the opportunistic pathogen Pseudomonas aeruginosa incorporates L-rhamnose both in the O-antigens of vari-Summary ous serotypes and in the common core polysaccharide of LPS of all strains. Mutants with impaired LPS synthe-dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) catalyzes the final step in the conversion of dTDP-sis lose their virulence and can be cleared by the immune system [5]. The widespread distribution of L-rhamnose D-glucose to dTDP-L-rhamnose in an NAD(P)H-and Mg 2؉ -dependent reaction. L-rhamnose biosynthesis is