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The metabolism of nitrosothiols in the Mycobacteria: identification and characterization of S-nitrosomycothiol reductase.
Biochem J 2003; 374(Pt 3):657-66BJ

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+Show Affiliations

Division of Chemical Pathology, Faculty of Health Sciences, University of Cape Town, Observatory 7935, South Africa.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12809551

Citation

Vogt, Ryan N., et al. "The Metabolism of Nitrosothiols in the Mycobacteria: Identification and Characterization of S-nitrosomycothiol Reductase." The Biochemical Journal, vol. 374, no. Pt 3, 2003, pp. 657-66.
Vogt RN, Steenkamp DJ, Zheng R, et al. The metabolism of nitrosothiols in the Mycobacteria: identification and characterization of S-nitrosomycothiol reductase. Biochem J. 2003;374(Pt 3):657-66.
Vogt, R. N., Steenkamp, D. J., Zheng, R., & Blanchard, J. S. (2003). The metabolism of nitrosothiols in the Mycobacteria: identification and characterization of S-nitrosomycothiol reductase. The Biochemical Journal, 374(Pt 3), pp. 657-66.
Vogt RN, et al. The Metabolism of Nitrosothiols in the Mycobacteria: Identification and Characterization of S-nitrosomycothiol Reductase. Biochem J. 2003 Sep 15;374(Pt 3):657-66. PubMed PMID: 12809551.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The metabolism of nitrosothiols in the Mycobacteria: identification and characterization of S-nitrosomycothiol reductase. AU - Vogt,Ryan N, AU - Steenkamp,Daniel J, AU - Zheng,Renjian, AU - Blanchard,John S, PY - 2003/06/17/accepted PY - 2003/05/30/revised PY - 2003/04/30/received PY - 2003/6/18/pubmed PY - 2003/10/1/medline PY - 2003/6/18/entrez SP - 657 EP - 66 JF - The Biochemical journal JO - Biochem. J. VL - 374 IS - Pt 3 N2 - 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. SN - 1470-8728 UR - https://www.unboundmedicine.com/medline/citation/12809551/The_metabolism_of_nitrosothiols_in_the_Mycobacteria:_identification_and_characterization_of_S_nitrosomycothiol_reductase_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20030642 DB - PRIME DP - Unbound Medicine ER -