The metabolism of nitrosothiols in the mycobacteria: Identification and characterization of S-nitrosomycothiol reductase

Ryan N. Vogt, Daniel J. Steenkamp, Renjian Zheng, John S. Blanchard

Research output: Contribution to journalArticle

50 Citations (Scopus)

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 38 263 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. 116 700 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.

Original languageEnglish (US)
Pages (from-to)657-665
Number of pages9
JournalBiochemical Journal
Volume374
Issue number3
DOIs
StatePublished - Sep 15 2003

Fingerprint

Mycobacterium smegmatis
Mycobacterium
Metabolism
Oxidoreductases
Mycobacterium tuberculosis
S-Nitrosoglutathione
NAD
Enzymes
Affinity chromatography
formic acid
glutathione-independent formaldehyde dehydrogenase
Affinity Chromatography
Genes
S-Nitrosothiols
Ion chromatography
Glutathione Disulfide
Molecular mass
Hydrophobic and Hydrophilic Interactions
Stoichiometry
Nitrates

Keywords

  • Formaldehyde dehydrogenase
  • Mycobacteria
  • Mycothiol
  • S-nitrosomycothiol reductase

ASJC Scopus subject areas

  • Biochemistry

Cite this

The metabolism of nitrosothiols in the mycobacteria : Identification and characterization of S-nitrosomycothiol reductase. / Vogt, Ryan N.; Steenkamp, Daniel J.; Zheng, Renjian; Blanchard, John S.

In: Biochemical Journal, Vol. 374, No. 3, 15.09.2003, p. 657-665.

Research output: Contribution to journalArticle

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