Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a molybdoenzyme that catalyses the reduction of the TMAO to trimethylamine (TMA) with a redox potential of +130 mV. We have successfully substituted the molybdenum with tungsten and obtained an active tungsto-TMAO reductase. Kinetic studies revealed that the catalytic efficiency of the tungsto-substituted TMAO reductase (W-TorA) was increased significantly (twofold), although a decrease of about 50% in its kcat was found compared with the molybdo-TMAO reductase (Mo-TorA). W-TorA is more sensitive to high pH, is less sensitive to high NaCl concentration and is more heat resistant than Mo-TorA. Most importantly, the W-TorA becomes capable of reducing sulphoxides and supports the anaerobic growth of a bacterial host on these substrates. The evolutionary implication and mechanistic significance of the tungsten substitution are discussed.
Mol Microbiol 1999 Apr
PMID:Enzymatic and physiological properties of the tungsten-substituted molybdenum TMAO reductase from Escherichia coli. 1021 69

Improved assays for the molybdenum enzyme dimethylsulfoxide reductase (DMSOR) with dimethyl sulfoxide (DMSO) and with dimethyl sulfide (DMS) as substrates are described. Maximum activity was observed at pH 6.5 and below and at 8.3, respectively. Rapid-scan stopped-flow spectrophotometry has been used to investigate the reduction of the enzyme by DMS to a species previously characterized by its UV-visible spectrum [McAlpine, A. S., McEwan, A. G., and Bailey, S. (1998) J. Mol. Biol. 275, 613-623], and its subsequent reoxidation by DMSO. Both these two-electron reactions were faster than enzyme turnover under steady-state conditions, indicating that one-electron reactions with artificial dyes were rate-limiting. Second-order rate constants for the two-electron reduction and reoxidation reactions at pH 5.5 were (1.9 +/- 0.1) x 10(5) and (4.3 +/- 0.3) x 10(2) M-1 s-1, respectively, while at pH 8.0, the catalytic step was rate-limiting (62 s-1). Kinetically, for the two-electron reactions, the enzyme is more effective in DMS oxidation than in DMSO reduction. Reduction of DMSOR by DMS was incomplete below approximately 1 mM DMS but complete at higher concentrations, implying that the enzyme's redox potential is slightly higher than that of the DMS-DMSO couple. In contrast, reoxidation of the DMS-reduced state by DMSO was always incomplete, regardless of the DMSO concentration. Evidence for the existence of a spectroscopically indistinguishable reduced state, which could not be reoxidized by DMSO, was obtained. Brief reaction (less than approximately 15 min) of DMS with DMSOR was fully reversible on removal of the DMS. However, in the presence of excess DMS, a further slow reaction occurred aerobically, but not anaerobically, to yield a stable enzyme form having a lambdamax at 660 mn. This state (DMSORmod) retained full activity in steady-state assays with DMSO, but was inactive toward DMS. It could however be reconverted to the original resting state by reduction with methyl viologen radical and reoxidation with DMSO. We suggest that in this enzyme form two of the dithiolene ligands of the molybdenum have dissociated and formed a disulfide. The implications of this new species are discussed in relation both to conflicting published information for DMSOR from X-ray crystallography and to previous spectroscopic data for its reduced forms.
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PMID:Reactions of dimethylsulfoxide reductase from Rhodobacter capsulatus with dimethyl sulfide and with dimethyl sulfoxide: complexities revealed by conventional and stopped-flow spectrophotometry. 1038 97

Molybdoenzymes are involved in a variety of essential pathways including nitrate assimilation, sulfur and/or purine metabolism and abscisic acid biosynthesis. Most organisms produce several such enzymes requiring a molybdopterin cofactor for catalytic function. Mutations that result in a lack of the molybdopterin cofactor display a pleiotropic loss of molybdoenzyme activities, and this phenotype has been used to identify genes involved in cofactor biosynthesis or utilization. Although several cofactor genes have been analyzed in prokaryotes, much less is known concerning eukaryotic molybdenum cofactor (MoCF) genes. This work is focused on the Drosophila MoCF gene cinnamon (cin) which encodes a multidomain protein, CIN, that shows significant similarity to three proteins encoded by separate prokaryotic MoCF genes. These domains are also present in the product of cnx1, an Arabidopsis MoCF gene, and in GEPHYRIN, a rat protein thought to organize the glycine receptor, GlyR, within the postsynaptic membrane. Since this apparent consolidation of separate prokaryotic genes into a single eukaryotic gene is a feature of other conserved metabolic pathways, we wished to determine whether the protein's function is also conserved. This report shows that the plant gene cnx1 can rescue both enzymatic and physiological defects of Drosophila carrying cin mutations, indicating that the two genes serve similar or identical functions. In addition, we have investigated the relationship between CINNAMON and GEPHYRIN, using immunohistochemical methods to localize the CIN protein in Drosophila embryos. Most of the CIN protein, like GEPHYRIN in the rat CNS, is localized to the cell borders and shows a tissue-specific pattern of expression. In a parallel study, antibody to GEPHYRIN revealed the same tissue-specific expression pattern in fly embryos. Both antibodies show altered staining patterns in cin mutants. Taken together, these results suggest that GEPHYRIN may also carry out a MoCF-related function.
Mol Gen Genet 1999 Jun
PMID:The Drosophila cinnamon gene is functionally homologous to Arabidopsis cnx1 and has a similar expression pattern to the mammalian gephyrin gene. 1039 4

The X-ray crystal structure of Klebsiella pneumoniae nitrogenase component 1 (Kp1) has been determined and refined to a resolution of 1.6 A, the highest resolution reported for any nitrogenase structure. Models derived from three 1.6 A resolution X-ray data sets are described; two represent distinct oxidation states, whilst the third appears to be a mixture of both oxidized and reduced states (or perhaps an intermediate state). The structures of the protein and the iron-molybdenum cofactor (FeMoco) appear to be largely unaffected by the redox status, although the movement of Ser beta90 and a surface helix in the beta subunit may be of functional significance. By contrast, the 8Fe-7S P-cluster undergoes discrete conformational changes involving the movement of two iron atoms. Comparisons with known component 1 structures reveal subtle differences in the FeMoco environment, which could account for the lower midpoint potential of this cluster in Kp1. Furthermore, a non-proline- cis peptide bond has been identified in the alpha subunit that may have a functional role. It is within 10 A of the FeMoco and may have been overlooked in other component 1 models. Finally, metal-metal and metal-sulphur distances within the metal clusters agree well with values derived from EXAFS studies, although they are generally longer than the values reported for the closely related protein from Azotobacter vinelandii. A number of bonds between the clusters and their ligands are distinctly longer than the EXAFS values, in particular, those involving the molybdenum atom of the FeMoco.
J Mol Biol 1999 Oct 01
PMID:New insights into structure-function relationships in nitrogenase: A 1.6 A resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein. 1052 12

At pH 2.8 and in the presence of 0.090% p-octylpolyethyleneglycol phenylether, the resonance light-scattering (RLS) spectrum of molybdenum(VI) complex with dibromohydroxyphenylfluorone (DBHPF) has a sharp peak at 586 nm. If the micro protein coexists with Mo(VI) and DBHPF, the RLS intensity of the complex at 586 nm is significantly enhanced by protein due to the binding interaction between protein and DBHPF-Mo(VI) complex. Based on this a new assay for protein is described. The dynamic ranges for bovine and human serum albumins are both 0.05-0.75 mg l-1 with detection limits of 13 and 15 ng ml-1, respectively. Besides high sensitivity, the method is characterized by good reproducibility, rapidity of reaction, good stability of chemical system, commonality of spectrofluorometer, few coexisting substances, especially detergents. The determinations of diluted human serum and urine by this method give the results very close to these by the Coomassie brilliant blue G-250 colorimetry, with relative standard deviations of five duplicates of 1.8-2.5%.
Spectrochim Acta A Mol Biomol Spectrosc 1999 Dec
PMID:A highly sensitive assay for protein using resonance light-scattering technique with dibromohydroxyphenylfluorone-molybdenum(VI) complex. 1065 51

The evolution and spread of insecticide resistance is an important factor in human disease prevention and crop protection. The mosquito Culex quinquefasciatus is the main vector of the disease filariasis and a member of a species complex which is a common biting nuisance worldwide. The common insecticide resistance mechanism in this species involves germline amplification of the esterases estalpha21 and estbeta21. This amplification has arisen once and rapidly spread worldwide. Less common and more variable resistance phenotypes involve coamplification of estalpha3 and estbeta1, or individual amplification of a single estbeta1, different alleles of the same estalpha and estbeta gene loci. Estalpha21 and estbeta21 are on the same large fragment of amplified DNA (amplicon) 2.7 kb apart. We have now shown that this amplicon contains another full-length gene immediately 5' of estalpha21 which codes for a molybdenum-containing hydroxylase, with highest homology to aldehyde oxidase (AO) from other organisms. The full-length putative AO gene is not present on the estalpha3/estbeta1 or estbeta1 amplicons, but multiple truncated 5' ends of this gene are present around the presumed estalpha3/estbeta1 amplicon breakpoint. Polymerase chain reaction (PCR) analysis of insecticide-susceptible genomic DNA demonstrated that a different allele of the putative AO gene in its non-amplified form is immediately 5' of estalpha. The 'AO' gene on the estalpha21/estbeta21 amplicon is expressed and resistant insects have greater AO activity. This AO activity is sensitive to inhibition by an aldehyde-containing herbicide and pesticide. This enzyme may confer a selective advantage to these insects in the presence of insecticide, as AO in mammals is believed to be important in the detoxification process of several environmental pollutants.
Insect Mol Biol 2000 Feb
PMID:Aldehyde oxidase is coamplified with the World's most common Culex mosquito insecticide resistance-associated esterases. 1067 76

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 specificity 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 and c=177.1 A, and diffraction data were obtained to beyond 2.8 A. 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 reflected in the electrostatic potential surfaces of both homologous enzymes, one exception being the surface potential in a region identifiable 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 confirmation 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.
J Mol Biol 2000 Mar 17
PMID:Gene sequence and crystal structure of the aldehyde oxidoreductase from Desulfovibrio desulfuricans ATCC 27774. 1070 12

DMSO reductase (DMSOR) from Rhodobacter capsulatus, well-characterised as a molybdoenzyme, will bind tungsten. Protein crystallography has shown that tungsten in W-DMSOR is ligated by the dithiolene group of the two pyranopterins, the oxygen atom of Ser147 plus another oxygen atom, and is located in a very similar site to that of molybdenum in Mo-DMSOR. These conclusions are consistent with W L(III)-edge X-ray absorption, EPR and UV/visible spectroscopic data. W-DMSOR is significantly more active than Mo-DMSOR in catalysing the reduction of DMSO but, in contrast to the latter, shows no significant ability to catalyse the oxidation of DMS.
J Mol Biol 2000 Jun 09
PMID:Dimethylsulfoxide reductase: an enzyme capable of catalysis with either molybdenum or tungsten at the active site. 1083 70

Crystal structures of carbon monoxide dehydrogenase (CODH), a seleno-molybdo-iron-sulfur flavoprotein from the aerobic carbon monoxide utilizing carboxidotrophic eubacterium Hydrogenophaga pseudoflava, have been determined from the enzyme synthesized at high (Mo(plus) CODH) and low intracellular molybdenum content (Mo(minus) CODH) at 2.25 A and 2.35 A resolution, respectively. The structures were solved by Patterson search methods utilizing the enzyme from Oligotropha carboxidovorans as the initial model. The CODHs from both sources are structurally very much conserved and show the same overall fold, architecture and arrangements of the molybdopterin-cytosine dinucleotide-type of molybdenum cofactor, the type I and type II [2Fe-2S] clusters and the flavin-adenine dinucleotide. Unlike the CODH from O. carboxidovorans, the enzyme from H. pseudoflava reveals a unique post-translationally modified C(gamma)-hydroxy-Arg384 residue which precedes the catalytically essential S-selanyl-Cys385 in the active-site loop. In addition, the Trp193 which shields the isoalloxazine ring of the flavin-adenine dinucleotide in the M subunit of the H. pseudoflava CODH is a Tyr193 in the O. carboxidovorans CODH. The hydrogen bonding interaction pattern of the molybdenum cofactor involves 27 hydrogen bonds with the surrounding protein. Of these, eight are with the cytosine moiety, eight with the pyrophosphate, six with the pyranopterin, and five with the ligands of the Mo ion. The structure of the catalytically inactive Mo(minus) CODH indicates that an intracellular Mo-deficiency affects exclusively the active site of the enzyme as an incomplete non-functional molybdenum cofactor was synthesized. The 5'-CDP residue was present in Mo(minus) CODH, whereas the Mo-pyranopterin moiety was absent. In Mo(plus) CODH the selenium faces the Mo ion and flips away from the Mo site in Mo(minus) CODH. The different side-chain conformations of the active-site residues S-selanyl-Cys385 and Glu757 in Mo(plus) and Mo(minus) CODH indicate a side-chain flexibility and a function of the Mo ion in the proper orientation of both residues.
J Mol Biol 2000 Sep 01
PMID:The effect of intracellular molybdenum in Hydrogenophaga pseudoflava on the crystallographic structure of the seleno-molybdo-iron-sulfur flavoenzyme carbon monoxide dehydrogenase. 1096 17

The molybdopterin cofactor (MoCF) is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the MoCF to have a terminal, cyanolysable sulphur ligand. In the sulphite oxidase/nitrate reductase class, an oxygen is present in the same position. Mutations in both the ma-l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans result in loss of activities of all molybdoenzymes that necessitate a cyanolysable sulphur in the active centre. The ma-l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate-dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulphur donor. Key similarities were found with NifS, the enzyme involved in the generation of the iron-sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum-bound sulphur ligand. We have identified putative homologues of these genes in a variety of organisms, including humans. The human homologue is located in chromosome 18.q12.
Mol Microbiol 2000 Oct
PMID:Comparison of the sequences of the Aspergillus nidulans hxB and Drosophila melanogaster ma-l genes with nifS from Azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulphur atom in the molybdopterin cofactor. 1102 94


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