title: The metabolism of nitrosothiols in the Mycobacteria: identification and characterization of S-nitrosomycothiol reductase.
abstract: When grown in culture Mycobacterium smegmatis metabolized S-nitrosoglutathione to oxidized glutathione and nitrate, which suggested a possible involvement of an S-nitrosothiol reductase and mycobacterial haemoglobin. The mycothiol-dependent formaldehyde dehydrogenase from M. smegmatis was purified by a combination of Ni2+-IMAC (immobilized metal ion affinity chromatography), hydrophobic interaction, anion-exchange and affinity chromatography. The enzyme had a subunit molecular mass of 38263 kDa. Steady-state kinetic studies indicated that the enzyme catalyses the NAD+-dependent conversion of S-hydroxymethylmycothiol into formic acid and mycothiol by a rapid-equilibrium ordered mechanism. The enzyme also catalysed an NADH-dependent decomposition of S-nitrosomycothiol (MSNO) by a sequential mechanism and with an equimolar stoichiometry of NADH:MSNO, which indicated that the enzyme reduces the nitroso group to the oxidation level of nitroxyl. Vmax for the MSNO reductase reaction indicated a turnover per subunit of approx. 116700 min(-1), which was 76-fold faster than the formaldehyde dehydrogenase activity. A gene, Rv2259, annotated as a class III alcohol dehydrogenase in the Mycobacterium tuberculosis genome was cloned and expressed in M. smegmatis as the C-terminally His6-tagged product. The purified recombinant enzyme from M. tuberculosis also catalysed both activities. M. smegmatis S-nitrosomycothiol reductase converted MSNO into the N -hydroxysulphenamide, which readily rearranged to mycothiolsulphinamide. In the presence of MSNO reductase, M. tuberculosis HbN (haemoglobin N) was converted with low efficiency into metHbN [HbN(Fe3+)] and this conversion was dependent on turnover of MSNO reductase. These observations suggest a possible route in vivo for the dissimilation of S-nitrosoglutathione.
authors.affiliation: Division of Chemical Pathology, Faculty of Health Sciences, University of Cape Town, Observatory 7935, South Africa.
authors.forename: Ryan N
authors.initials: RN
authors.lastname: Vogt
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Steenkamp
authors.forename: Renjian
authors.initials: R
authors.lastname: Zheng
authors.forename: John S
authors.initials: JS
authors.lastname: Blanchard
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D004187:Disaccharides / Q000378:metabolism*; D004795:Enzyme Stability; D006020:Glycopeptides; D006863:Hydrogen-Ion Concentration; D007294:Inositol; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011720:Pyrazoles / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D026422:S-Nitrosoglutathione / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; D006020:Glycopeptides; D011720:Pyrazoles; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C089265:mycothiol; D007294:Inositol; D026422:S-Nitrosoglutathione; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent; D009247:NADH, NADPH Oxidoreductases; D003545:Cysteine