Gene sequence and crystal structure of the aldehyde oxidoreductase from Desulfovibrio desulfuricans ATCC 277741 (original) (raw)
Related papers
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.
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. As deduced from this work, both these enzymes are very closely related in terms of their sequences as well as 3D structures. The comparison allowed con®rmation and establishment of features that are essential for their function; namely, conserved residues in the active-site, catalytically relevant water molecules and recognition of the physiological electron acceptor docking site.
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.
European Journal of Biochemistry, 1993
The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe-2S] centers. The enzyme was characterized by SDSPAGE, gel-filtration and analytical ultracentrifugation experiments. It was crystallized at !"C, pH 7.2, using isopropanol and MgC1, as precipitants. The crystals diffract beyond 0.3-nm (3.0-A) resolution and belong to space group P6,22 or its enantiomorph, with cell dimensions u = b = 14.45 nm and c = 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 X nm'/Da (2.5 A3/Da), consistent with the experimental crystal density, p = 1.14 g/cm3. One dimer (approximately 2 X 100 kDa) is located on a crystallographic twofold axis.
Structure and function of molybdopterin containing enzymes
Progress in Biophysics and Molecular Biology, 1997
ÐMolybdopterin containing enzymes are present in a wide range of living systems and have been known for several decades. However, only in the past two years have the ®rst crystal structures been reported for this type of enzyme. This has represented a major breakthrough in this ®eld. The enzymes share common structural features, but reveal dierent polypeptide folding topologies. In this review we give an account of the related spectroscopic information and the crystallographic results, with emphasis on structure-function studies. # 1998 Elsevier Science Ltd. All rights reserved CONTENTS I. INTRODUCTION II. CLASSIFICATION OF MOLYBDOPTERIN CONTAINING ENZYMES 2.1. The xanthine oxidase family 2.2. The DMSO reductase family 2.3. The sul®te oxidase family III. THE DIVERSITY OF THE MOLYBDOPTERIN COFACTOR IV. STRUCTURE-FUNCTION STUDIES 4.1. Crystal structure data for the xanthine oxidase family of enzymes 4.1.1. Three-dimensional structure of the aldehyde oxidoreductase from Desulfovibrio gigas 4.1.2. Mechanistic implications 4.2. The DMSO reductases 4.2.1. Three-dimensional structure of DMSOR from Rhodobacter sphaeroides and Rhodobacter capsulatus 4.2.2. Structure of the metal site and mechanistic implications 4.3.
Structure, 2002
Gene Sequence and the 1.8 Å Crystal Structure of the Tungsten-Containing Formate Dehydrogenase from Desulfovibrio gigas dehydrogenase isolated from D. desulfuricans ATCC 27774 is homologous to DvMo-FDH and was characterized by EPR and Mö ssbauer spectroscopies [4]. A monoheme cytochrome, c 553 , was identified as the physiological partner for DvMo-FDH [3, 5]. The physiological donor of DgW-FDH is also a monoheme cytochrome that was recently purified (our unpublished data). Departamento de Química FCT DgW-FDH consists of two subunits of 977 and 214 amino acids, and it belongs to the DMSO reductase Universidade Nova de Lisboa 2829-516 Caparica (DMSOR) family of enzymes, one of the four classes into which molybdopterin-containing enzymes have been Portugal 2 Max-Planck-Institut fü r Biochemie classified [6-9]. This family is considerably broad, and, besides DMSOR [10, 11], it includes enzymes such as Am Klopferspitz 18a D-82152 Martinsried dissimilatory nitrate reductases (NAP) [12] and formate dehydrogenases. The members of this family have two Germany molybdopterins (MGD cofactor) in the coordination sphere of Mo (or W), which is also bound to an amino acid side chain. This amino acid changes with the enzy-Summary matic functionality and is a serine residue in DMSOR, a cysteine in NAP, and a selenocysteine in FDHs [2, 13].
The molybdenum iron-sulphur protein from Desulfovibrio gigas as a form of aldehyde oxidase
Biochemical Journal, 1987
The molybdenum iron-sulphur protein originally isolated from Desulfovibrio gigas by Moura, Xavier, Bruschi, Le Gall, Hall & Cammack [(1976) Biochem. Biophys. Res. Commun. 72, 782-789] has been further investigated by e.p.r. spectroscopy of molybdenum(V). The signal obtained on extended reduction of the protein with sodium dithionite has been shown, by studies at 9 and 35 HGz in 1H2O and 2H2O and computer simulations, to have parameters corresponding to those of the Slow signal from the inactive desulpho form of various molybdenum-containing hydroxylases. Another signal obtained on brief reduction of the protein with small amounts of dithionite was shown by e.p.r. difference techniques to be a Rapid type 2 signal, like that from the active form of such enzymes. In confirmation that the protein is a molybdenum-containing hydroxylase, activity measurements revealed that it had aldehyde:2,6-dichlorophenol-indophenol oxidoreductase activity. No such activity towards xanthine or purine wa...