Publications

• Purification and properties of trimethylamine N-oxide reductase from aerobic photosynthetic bacterium Roseobacter denitrificans.

  Arata H., Shimizu M., Takamiya K.

  Trimethylamine N-oxide (TMAO) reductase was purified from an aerobic photosynthetic bacterium Roseobacter denitrificans. The enzyme was purified from cell-free extract by ammonium sulfate fractionation, DEAE ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified ... >> More

  Trimethylamine N-oxide (TMAO) reductase was purified from an aerobic photosynthetic bacterium Roseobacter denitrificans. The enzyme was purified from cell-free extract by ammonium sulfate fractionation, DEAE ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme was composed of two identical subunits with molecular weight of 90,000, as identified by SDS-polyacrylamide gel electrophoresis, containing heme c and a molybdenum cofactor. The molecular weight of the native enzyme determined by gel filtration was 172,000. The midpoint redox potential of heme c was +200 mV at pH 7.5. Absorption maxima appeared at 418,524, and 554 nm in the reduced state and 410 nm in the oxidized state. The enzyme reduced TMAO, nicotine acid N-oxide, picoline N-oxide, hydroxylamine, and bromate, but not dimethyl sulfoxide, methionine sulfoxide, chlorate, nitrate, or thiosulfate. Cytochrome c2 served as a direct electron donor. It probably catalyzes the electron transfer from cytochrome b-c1 complex to TMAO reductase. Cytochrome c552, another soluble low-molecular-weight cytochrome of this bacterium, also donated electrons directly to TMAO reductase. << Less

  J Biochem 112:470-475(1992) [PubMed] [EuropePMC]

• Isolation, cloning, sequence analysis and X-ray structure of dimethyl sulfoxide/trimethylamine N-oxide reductase from Rhodobacter capsulatus.

  Knablein J., Dobbek H., Ehlert S., Schneider F.

  The periplasmic enzyme dimethyl sulfoxide/trimethylamine N-oxide reductase (DMSOR/TMAOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimet ... >> More

  The periplasmic enzyme dimethyl sulfoxide/trimethylamine N-oxide reductase (DMSOR/TMAOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO). At a molybdenum redox centre, two single electrons are transferred from cytochrome c556 to the substrate, e.g. DMSO, generating dimethyl sulfide (DMS) and water. The operon encoding this enzyme was isolated, cloned and sequenced, and its chromosomal location determined. It was shown by analytical and crystallographic data that DMSOR and TMAOR are identical enzymes. Degenerate primers were derived from short peptide sequences and a 700 bp fragment was amplified by nested PCR, subsequently cloned and radioactively labeled to screen a prepared lambda DASH library. Positive lambda clones were subcloned into pBluescript and subsequently transformed into Escherichia coli to sequence the DMSOR/TMAOR operon. By an optimized protein purification high yields (5 mg protein/l culture) with a specific activity of 30 U/mg were obtained. The molecular mass was experimentally determined by electrospray mass spectroscopy (MS) to be 85034 Da and from the deduced amino acid sequence of the apoenzyme to be 85033 Da. The enzyme was crystallized in space group P4(1)2(1)2 with unit cell dimensions of a = b = 80.7 A and c = 229.2 A diffracting beyond 1.8 A. The three-dimensional structure was solved by a combination of multiple isomorphous replacement (MIR) and molecular replacement techniques. The atomic model was refined to an R-factor of 0.169 for 57394 independent reflections. The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center. The sole bis(molybdopterin guanine dinucleotide)molybdenum cofactor (1541 Da) of the single chain protein has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide (MGD) molecule. In addition, two oxo ligands and the oxygen of a serine side chain are bound to the molybdenum ion. << Less

  Biol Chem 378:293-302(1997) [PubMed] [EuropePMC]

• Crystal structure of oxidized trimethylamine N-oxide reductase from Shewanella massilia at 2.5-A resolution.

  Czjzek M., Dos Santos J.P., Pommier J., Giordano G., Mejean V., Haser R.

  The periplasmic trimethylamine N-oxide (TMAO) reductase from the marine bacteria Shewanella massilia is involved in a respiratory chain, having trimethylamine N-oxide as terminal electron acceptor. This molybdoenzyme belongs to the dimethyl sulfoxide (DMSO) reductase family, but has a different su ... >> More

  The periplasmic trimethylamine N-oxide (TMAO) reductase from the marine bacteria Shewanella massilia is involved in a respiratory chain, having trimethylamine N-oxide as terminal electron acceptor. This molybdoenzyme belongs to the dimethyl sulfoxide (DMSO) reductase family, but has a different substrate specificity than its homologous enzyme. While the DMSO reductases reduce a broad spectra of organic S-oxide and N-oxide compounds, TMAO reductase from Shewanella massilia reduces only TMAO as the natural compound. The crystal structure was solved by molecular replacement with the coordinates of the DMSO reductase from Rhodobacter sphaeroides. The overall fold of the protein structure is essentially the same as the DMSO reductase structures, organized into four domains. The molybdenum coordination sphere is closest to that described in the DMSO reductase of Rhodobacter capsulatus. The structural differences found in the protein environment of the active site could be related to the differences in substrate specificity of these enzymes. In close vicinity of the molybdenum ion a tyrosine residue is missing in the TMAO reductase, leaving a greater space accessible to the solvent. This tyrosine residue has contacts to the oxo groups in the DMSO reductase structures. The arrangement and number of charged residues lining the inner surface of the funnel-like entrance to the active site, is different in the TMAO reductase than in the DMSO reductases from Rhodobacter species. Furthermore a surface loop at the top of the active-site funnel, for which no density was present in the DMSO reductase structures, is well defined in the oxidized form of the TMAO reductase structure, and is located on the border of the funnel-like entrance of the active center. << Less

  J. Mol. Biol. 284:435-447(1998) [PubMed] [EuropePMC]

• Electron transfer and binding of the c-type cytochrome TorC to the trimethylamine N-oxide reductase in Escherichia coli.

  Gon S., Giudici-Orticoni M.-T., Mejean V., Iobbi-Nivol C.

  Reduction of trimethylamine N-oxide (E'(0(TMAO/TMA)) = +130 mV) in Escherichia coli is carried out by the Tor system, an electron transfer chain encoded by the torCAD operon and made up of the periplasmic terminal reductase TorA and the membrane-anchored pentahemic c-type cytochrome TorC. Although ... >> More

  Reduction of trimethylamine N-oxide (E'(0(TMAO/TMA)) = +130 mV) in Escherichia coli is carried out by the Tor system, an electron transfer chain encoded by the torCAD operon and made up of the periplasmic terminal reductase TorA and the membrane-anchored pentahemic c-type cytochrome TorC. Although the role of TorA in the reduction of trimethylamine N-oxide (TMAO) has been clearly established, no direct evidence for TorC involvement has been presented. TorC belongs to the NirT/NapC c-type cytochrome family based on homologies of its N-terminal tetrahemic domain (TorC(N)) to the cytochromes of this family, but TorC contains a C-terminal extension (TorC(C)) with an additional heme-binding site. In this study, we show that both domains are required for the anaerobic bacterial growth with TMAO. The intact TorC protein and its two domains, TorC(N) and TorC(C), were produced independently and purified for a biochemical characterization. The reduced form of TorC exhibited visible absorption maxima at 552, 523, and 417 nm. Mediated redox potentiometry of the heme centers of the purified components identified two negative midpoint potentials (-177 and -98 mV) localized in the tetrahemic TorC(N) and one positive midpoint potential (+120 mV) in the monohemic TorC(C). In agreement with these values, the in vitro reconstitution of electron transfer between TorC, TorC(N), or TorC(C) and TorA showed that only TorC and TorC(C) were capable of electron transfer to TorA. Surprisingly, interaction studies revealed that only TorC and TorC(N) strongly bind TorA. Therefore, TorC(C) directly transfers electrons to TorA, whereas TorC(N), which probably receives electrons from the menaquinone pool, is involved in both the electron transfer to TorC(C) and the binding to TorA. << Less

  J. Biol. Chem. 276:11545-11551(2001) [PubMed] [EuropePMC]