Reductases from Rhodobacter capsulatus

Knowledge of the enzymes used by microorganisms in the transformation of nitroaromatic compounds is limited. Blasco Castillo (1993) characterized an inducible nitrophenol reductase from Rhodobacter capsulatus that catalyzed the reduction of 2,4-dinitrophenol (DNP) to 2-amino-4-nitrophenol. This enzyme was a dimer that contained flavin mononucleotide and possibly nonheme iron as... 

Adams, B., Smith, A. T., Bailey, S., McEwan, A. G., and Bray, R. C., 1999, Reactions of dimethyl-sulfoxide reductase from Rhodobacter capsulatus with dimethylsulfide and with dimethyl-sulfoxide complexities revealed by conventional and stopped-flow spectrophotometry. 

McAlpine, A. S., McEwan, A. G., Shaw, A. L., and Bailey, S., 1997, Molybdenum active centre of DMSO reductase from Rhodobacter capsulatus. crystal stracture of the oxidized enzyme at 1.82 resolution and the dithionite reduced enzyme at 2.8 resolution, J. Biol. Inorg. Chem. 2 6909701. 

Schneider, F., Loewe, J., Huber, R., Schindelin, H., and Kisker, C., 1996, Crustal structure of dimethyl sulfoxide reductase from Rhodobacter capsulatus at 1.88 resolution, J. Mol. Biol 263 53n63. 

The periplasmic DMSO reductase from Rhodobacter capsulatus has also been studied by direct electrochemistry. When adsorbed on a pyrolytic graphite electrode, non-turnover signals were observed and assigned to the Mo and couples, there being no other redox active groups in the... 

Raman spectroscopy was employed to investigate the structure of the molybdenum cofactor in DMSO-reductase from Rhodobacter capsulatus. vMo = 0 was at 865 cm V ... 

A selection of biocatalytic deoxygenation reactions is shovm in Figure 1.8. The reducing power of baker s yeast in an ethanol-water mixture and sodium hydroxide at 60° C has been found effective for the rapid and selective reduction of a series of N-oxides like aromatic and heteroaromatic N-oxide compounds [118]. DMSO reductase from Rhodobacter sphaeroides f sp. denitrificans catalyzed the (S)-enantioselective reduction of various sulfoxides and enabled the resolution of racemic sulfoxides for the synthesis of (R)-sulfoxides with >97% ee [119,120]. Purified dimethyl sulfoxide reductase from Rhodobacter capsulatus resolved a racemic mixture of methyl p-tolyl sulfoxide by catalyzing the reduction of (S)-methyl p-tolyl sulfoxide and gave enantio-merically pure (J )-methyl p-tolyl sulfoxide in 88% yield, while whole cells of E. coli,... 

Hanlon, S.P., Graham, D.L, Hogan, P.J., Holt, RA., Reeve, C.D., Shad, A.L., and McEwan, A.G. (1998) Asymmetric reduction of racemic sulfoxides by dimethylsulfoxide reductases from Rhodobacter capsulatus, Escherichia coli and Proteus species. Microbiology, 144, 2247-2253. 

Shaw, A. L., Hochkoeppler, A., Bonora, P., Zannoni, D., Hanson, G., and McEwan, A. G., 1999, Characterization of DorC from Rhodobacter capsulatus, a c-type cytochrome involved in electron transfer to dimediylsulfoxide reductase, J. Biol. Chem. 274 9911fi9914. 

Recently Blasco and Castillo (4) reported the characterization of a nitrophenol nitroreductase purified from Rhodobacter capsulatus. It is a soluble, FMN-containing flavoprotein that uses NAD(P)H as electron donor, and reduces 2,4-dinitrophenol to 2-amino-4-nitrophenol which is not further metabolized (Fig. 4). The native enzyme is a homodimer of 54 kDa. The inhibition of enzymatic activity by metal chelators, salicylhy-droxamate, /7-hydroxymercuribenzoate and Cu indicates the importance of sulfhydryl groups in the stability and/or activity of the protein, and the possible involvement of non-heme iron in reductase activity. Nitrophenol reductase activity was induced by a variety of nitrophenols, but not by other nitroaromatic compounds. However, its induction was suppressed by the inclusion of ammonium in the medium, and its role in the cell is not clear. 

Total mass. Per subunit. Structural data can be obtained from the Brookhaven Protein Data Bank (PDB) at http //www.rcsb.org/pdb. Other Mo-MPT enzyme PDB codes include Hydrogenophagapseudoflava CO dehydrogenase, IFFV Desulfovibrio desulfuricans aldehyde oxidoreductase, IDGJ Rhodobacter capsulatus DMSO reductase, 4DMR R. sphaeroides nitrate reductase (dissimilatory), lOGY D. 

The ferroxidase center, important for rapid oxidation of Fe to Fe, was discovered relatively recently in the history of research into the metal sites in ferritins. Ferroxidase activity within H subunits appears to occur at a dinuclear site situated within a four-helix bundle and resembling the dinuclear centers found in ribonucleotide reductase, methane monooxygenase, fatty acid desaturases, and ruberythrin (Chapter 8.11). In bacterioferritins, for which protein crystal structures have been reported for ferritin from Escherichia col and Rhodobacter capsulatus the overall motif of a shell of 24 subunits with relative masses of about 18,500 Da is preserved but there are also 12 protoporphyrin IX heme groups present with unknown function which might have a role in connecting the dimer units and are buried within the shell between identical subunits related by twofold symmetry. In these bacterioferritins the subunits are all identical and contain both ftrroxidase and nucleation sites. 

There are three very well studied DMSO reductase enzymes. The enzymes isolated from the purple photosynthetic bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides (DorA) are periplasmic and share a high sequence identity. They are also the structurally simplest of all Mo enzymes ca. 85 kDa enzymes bearing a single redox active cofactor (the Mo active site). DMSO reductase from E. coli is a more complex membrane bound 140 kDa hetero-trimeric enzyme (DmsABC) bearing five Fe-S clusters in addition to the Mo active site. 

OCh 114 is capable of anaerobic respiration. Trimethyl amine N-oxide (TMAO) is one of the terminal oxidant (7). We have found tha the electron transport chain of the TMAO respiration of this bacterium is differenct from that of the related facultative phototrophs at two points (7) cytochrome hrSji complex is probably involved in OCh 114 while it is bypassed in Rhodobacter capsulatus (8) TMAO reductase of OCh 114 does not reduce dimethylsulfoxide while a single enzyme reduces both TMAO and dimethylsulphoxide in Rhodobacter sphaeroides (9) and R. capsulatas (10). 

However, some of these structures show unusual features and a crowded active site. EXAFS studies of Rhodobacter capsulatus DM SO reductase show the expected four Mo-S ligands and one Mo-O bond arising from a serine residue plus one Mo=0 in the reduced Mo (IV) state of the active site. This oxo ligand is removed upon oxidation of the metal ion to Mo(VI). In the oxidized structme, an aquo ligand is postulated to coordinate the molybdenum ion (Fig. 11.17c) [126-128]. An equivalent restdt has been reported for BSO that catalyzes the reduction of D-biotin D-sulfoxide to D-biotin [129]. A similar overall catalytic mechanism is expected for nitrate reductases (Fig. 11.18), which catalyze the following reaction ... 

Blasco, R., and F. Castillo. 1993. Characterization of a nitrophenol reductase from the phototrophic bacterium Rhodobacter capsulatus EIFI. Appl. Environ. Microbiol. 59 1774-1778. 

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