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.
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September 2003
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Research Article|
September 15 2003
The metabolism of nitrosothiols in the mycobacteria: identification and characterization of S-nitrosomycothiol reductase
Ryan N. VOGT;
Ryan N. VOGT
∗Division of Chemical Pathology, Faculty of Health Sciences, University of Cape Town, Observatory 7935, South Africa
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Daniel J. STEENKAMP;
Daniel J. STEENKAMP
1
∗Division of Chemical Pathology, Faculty of Health Sciences, University of Cape Town, Observatory 7935, South Africa
1To whom correspondence should be addressed (e-mail daan@chempath.uct.ac.za).
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Renjian ZHENG;
Renjian ZHENG
†Department of Biochemistry, Albert Einstein College of Medicine, The Bronx, NY 10461, New York, U.S.A.
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John S. BLANCHARD
John S. BLANCHARD
†Department of Biochemistry, Albert Einstein College of Medicine, The Bronx, NY 10461, New York, U.S.A.
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Publisher: Portland Press Ltd
Received:
April 30 2003
Revision Received:
May 30 2003
Accepted:
June 17 2003
Accepted Manuscript online:
June 17 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
The Biochemical Society, London © 2003
Biochem J (2003) 374 (3): 657–666.
Article history
Received:
April 30 2003
Revision Received:
May 30 2003
Accepted:
June 17 2003
Accepted Manuscript online:
June 17 2003
Citation
Ryan N. VOGT, Daniel J. STEENKAMP, Renjian ZHENG, John S. BLANCHARD; The metabolism of nitrosothiols in the mycobacteria: identification and characterization of S-nitrosomycothiol reductase. Biochem J 15 September 2003; 374 (3): 657–666. doi: https://doi.org/10.1042/bj20030642
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