Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Sep 15;374(Pt 3):657–666. doi: 10.1042/BJ20030642

The metabolism of nitrosothiols in the Mycobacteria: identification and characterization of S-nitrosomycothiol reductase.

Ryan N Vogt 1, Daniel J Steenkamp 1, Renjian Zheng 1, John S Blanchard 1
PMCID: PMC1223637  PMID: 12809551

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.

Full Text

The Full Text of this article is available as a PDF (197.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aleryani S., Milo E., Rose Y., Kostka P. Superoxide-mediated decomposition of biological S-nitrosothiols. J Biol Chem. 1998 Mar 13;273(11):6041–6045. doi: 10.1074/jbc.273.11.6041. [DOI] [PubMed] [Google Scholar]
  2. Bornemann C., Jardine M. A., Spies H. S., Steenkamp D. J. Biosynthesis of mycothiol: elucidation of the sequence of steps in Mycobacterium smegmatis. Biochem J. 1997 Aug 1;325(Pt 3):623–629. doi: 10.1042/bj3250623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Chan E. D., Chan J., Schluger N. W. What is the role of nitric oxide in murine and human host defense against tuberculosis?Current knowledge. Am J Respir Cell Mol Biol. 2001 Nov;25(5):606–612. doi: 10.1165/ajrcmb.25.5.4487. [DOI] [PubMed] [Google Scholar]
  5. Clancy R. M., Levartovsky D., Leszczynska-Piziak J., Yegudin J., Abramson S. B. Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as a bioactive intermediary. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3680–3684. doi: 10.1073/pnas.91.9.3680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Couture M., Guertin M. Purification and spectroscopic characterization of a recombinant chloroplastic hemoglobin from the green unicellular alga Chlamydomonas eugametos. Eur J Biochem. 1996 Dec 15;242(3):779–787. doi: 10.1111/j.1432-1033.1996.0779r.x. [DOI] [PubMed] [Google Scholar]
  7. Couture M., Yeh S. R., Wittenberg B. A., Wittenberg J. B., Ouellet Y., Rousseau D. L., Guertin M. A cooperative oxygen-binding hemoglobin from Mycobacterium tuberculosis. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11223–11228. doi: 10.1073/pnas.96.20.11223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crawford M. J., Goldberg D. E. Role for the Salmonella flavohemoglobin in protection from nitric oxide. J Biol Chem. 1998 May 15;273(20):12543–12547. doi: 10.1074/jbc.273.20.12543. [DOI] [PubMed] [Google Scholar]
  9. Di Cera E., Doyle M. L., Gill S. J. Alkaline Bohr effect of human hemoglobin Ao. J Mol Biol. 1988 Apr 5;200(3):593–599. doi: 10.1016/0022-2836(88)90545-1. [DOI] [PubMed] [Google Scholar]
  10. Frey Alexander D., Farrés Judith, Bollinger Christian J. T., Kallio Pauli T. Bacterial hemoglobins and flavohemoglobins for alleviation of nitrosative stress in Escherichia coli. Appl Environ Microbiol. 2002 Oct;68(10):4835–4840. doi: 10.1128/AEM.68.10.4835-4840.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gardner P. R., Gardner A. M., Martin L. A., Salzman A. L. Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10378–10383. doi: 10.1073/pnas.95.18.10378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gordge M. P., Addis P., Noronha-Dutra A. A., Hothersall J. S. Cell-mediated biotransformation of S-nitrosoglutathione. Biochem Pharmacol. 1998 Mar 1;55(5):657–665. doi: 10.1016/s0006-2952(97)00498-x. [DOI] [PubMed] [Google Scholar]
  13. Grassetti D. R., Murray J. F., Jr Determination of sulfhydryl groups with 2,2'- or 4,4'-dithiodipyridine. Arch Biochem Biophys. 1967 Mar;119(1):41–49. doi: 10.1016/0003-9861(67)90426-2. [DOI] [PubMed] [Google Scholar]
  14. Green R. M., Seth A., Connell N. D. A peptide permease mutant of Mycobacterium bovis BCG resistant to the toxic peptides glutathione and S-nitrosoglutathione. Infect Immun. 2000 Feb;68(2):429–436. doi: 10.1128/iai.68.2.429-436.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hausladen A., Gow A., Stamler J. S. Flavohemoglobin denitrosylase catalyzes the reaction of a nitroxyl equivalent with molecular oxygen. Proc Natl Acad Sci U S A. 2001 Aug 21;98(18):10108–10112. doi: 10.1073/pnas.181199698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jensen D. E., Belka G. K., Du Bois G. C. S-Nitrosoglutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme. Biochem J. 1998 Apr 15;331(Pt 2):659–668. doi: 10.1042/bj3310659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jensen D. E., Belka G. K., Dworkin C. Denitrosation of 1,3-dimethyl-2-cyano-1-nitrosoguanidine in rat primary hepatocyte cultures. Biochem Pharmacol. 1997 May 9;53(9):1297–1306. doi: 10.1016/s0006-2952(96)00861-1. [DOI] [PubMed] [Google Scholar]
  18. Jensen D. E., Belka G. K. Enzymic denitrosation of 1,3-dimethyl-2-cyano-1-nitrosoguanidine in rat liver cytosol and the fate of the immediate product S-nitrosoglutathione. Biochem Pharmacol. 1997 May 9;53(9):1279–1295. doi: 10.1016/s0006-2952(96)00860-x. [DOI] [PubMed] [Google Scholar]
  19. Kharitonov V. G., Sundquist A. R., Sharma V. S. Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen. J Biol Chem. 1995 Nov 24;270(47):28158–28164. doi: 10.1074/jbc.270.47.28158. [DOI] [PubMed] [Google Scholar]
  20. Kirsch Michael, de Groot Herbert. Formation of peroxynitrite from reaction of nitroxyl anion with molecular oxygen. J Biol Chem. 2002 Jan 17;277(16):13379–13388. doi: 10.1074/jbc.M108079200. [DOI] [PubMed] [Google Scholar]
  21. Kissner Reinhard, Koppenol Willem H. Product distribution of peroxynitrite decay as a function of pH, temperature, and concentration. J Am Chem Soc. 2002 Jan 16;124(2):234–239. doi: 10.1021/ja010497s. [DOI] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Liu L., Hausladen A., Zeng M., Que L., Heitman J., Stamler J. S. A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans. Nature. 2001 Mar 22;410(6827):490–494. doi: 10.1038/35068596. [DOI] [PubMed] [Google Scholar]
  24. Misset-Smits M., van Ophem P. W., Sakuda S., Duine J. A. Mycothiol, 1-O-(2'-[N-acetyl-L-cysteinyl]amido-2'-deoxy-alpha-D-glucopyranosyl)-D- myo-inositol, is the factor of NAD/factor-dependent formaldehyde dehydrogenase. FEBS Lett. 1997 Jun 9;409(2):221–222. doi: 10.1016/s0014-5793(97)00510-3. [DOI] [PubMed] [Google Scholar]
  25. Murphy M. E., Noack E. Nitric oxide assay using hemoglobin method. Methods Enzymol. 1994;233:240–250. doi: 10.1016/s0076-6879(94)33027-1. [DOI] [PubMed] [Google Scholar]
  26. Nakamura M., Nakamura S. Conversion of metmyoglobin to NO myoglobin in the presence of nitrite and reductants. Biochim Biophys Acta. 1996 Apr 17;1289(3):329–335. doi: 10.1016/0304-4165(95)00161-1. [DOI] [PubMed] [Google Scholar]
  27. Nathan C., Shiloh M. U. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):8841–8848. doi: 10.1073/pnas.97.16.8841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Newton G. L., Arnold K., Price M. S., Sherrill C., Delcardayre S. B., Aharonowitz Y., Cohen G., Davies J., Fahey R. C., Davis C. Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J Bacteriol. 1996 Apr;178(7):1990–1995. doi: 10.1128/jb.178.7.1990-1995.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Newton G. L., Av-Gay Y., Fahey R. C. A novel mycothiol-dependent detoxification pathway in mycobacteria involving mycothiol S-conjugate amidase. Biochemistry. 2000 Sep 5;39(35):10739–10746. doi: 10.1021/bi000356n. [DOI] [PubMed] [Google Scholar]
  30. Newton G. L., Unson M. D., Anderberg S. J., Aguilera J. A., Oh N. N., delCardayre S. B., Av-Gay Y., Fahey R. C. Characterization of Mycobacterium smegmatis mutants defective in 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside and mycothiol biosynthesis. Biochem Biophys Res Commun. 1999 Feb 16;255(2):239–244. doi: 10.1006/bbrc.1999.0156. [DOI] [PubMed] [Google Scholar]
  31. Norin A., Van Ophem P. W., Piersma S. R., Persson B., Duine J. A., Jörnvall H. Mycothiol-dependent formaldehyde dehydrogenase, a prokaryotic medium-chain dehydrogenase/reductase, phylogenetically links different eukaroytic alcohol dehydrogenases--primary structure, conformational modelling and functional correlations. Eur J Biochem. 1997 Sep 1;248(2):282–289. doi: 10.1111/j.1432-1033.1997.00282.x. [DOI] [PubMed] [Google Scholar]
  32. Ouellet Hugues, Ouellet Yannick, Richard Christian, Labarre Marie, Wittenberg Beatrice, Wittenberg Jonathan, Guertin Michel. Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide. Proc Natl Acad Sci U S A. 2002 Apr 16;99(9):5902–5907. doi: 10.1073/pnas.092017799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ouellet Hugues, Ouellet Yannick, Richard Christian, Labarre Marie, Wittenberg Beatrice, Wittenberg Jonathan, Guertin Michel. Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide. Proc Natl Acad Sci U S A. 2002 Apr 16;99(9):5902–5907. doi: 10.1073/pnas.092017799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pathania Ranjana, Navani Naveen K., Gardner Anne M., Gardner Paul R., Dikshit Kanak L. Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coli. Mol Microbiol. 2002 Sep;45(5):1303–1314. doi: 10.1046/j.1365-2958.2002.03095.x. [DOI] [PubMed] [Google Scholar]
  35. Poole R. K., Hughes M. N. New functions for the ancient globin family: bacterial responses to nitric oxide and nitrosative stress. Mol Microbiol. 2000 May;36(4):775–783. doi: 10.1046/j.1365-2958.2000.01889.x. [DOI] [PubMed] [Google Scholar]
  36. Rawat Mamta, Newton Gerald L., Ko Mary, Martinez Gladys J., Fahey Robert C., Av-Gay Yossef. Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals, and antibiotics. Antimicrob Agents Chemother. 2002 Nov;46(11):3348–3355. doi: 10.1128/AAC.46.11.3348-3355.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sanghani P. C., Stone C. L., Ray B. D., Pindel E. V., Hurley T. D., Bosron W. F. Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase. Biochemistry. 2000 Sep 5;39(35):10720–10729. doi: 10.1021/bi9929711. [DOI] [PubMed] [Google Scholar]
  38. Shirota F. N., Goon D. J., DeMaster E. G., Nagasawa H. T. Nitrosyl cyanide, a putative metabolic oxidation product of the alcohol-deterrent agent cyanamide. Biochem Pharmacol. 1996 Jul 12;52(1):141–147. doi: 10.1016/0006-2952(96)00174-8. [DOI] [PubMed] [Google Scholar]
  39. Spies H. S., Steenkamp D. J. Thiols of intracellular pathogens. Identification of ovothiol A in Leishmania donovani and structural analysis of a novel thiol from Mycobacterium bovis. Eur J Biochem. 1994 Aug 15;224(1):203–213. doi: 10.1111/j.1432-1033.1994.tb20013.x. [DOI] [PubMed] [Google Scholar]
  40. Steenkamp D. J., Husain M. The effect of tetrahydrofolate on the reduction of electron transfer flavoprotein by sarcosine and dimethylglycine dehydrogenases. Biochem J. 1982 Jun 1;203(3):707–715. doi: 10.1042/bj2030707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Venketaraman Vishwanath, Dayaram Yaswant K., Amin Amol G., Ngo Richard, Green Renee M., Talaue Meliza T., Mann Jessica, Connell Nancy D. Role of glutathione in macrophage control of mycobacteria. Infect Immun. 2003 Apr;71(4):1864–1871. doi: 10.1128/IAI.71.4.1864-1871.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wittenberg Jonathan B., Bolognesi Martino, Wittenberg Beatrice A., Guertin Michel. Truncated hemoglobins: a new family of hemoglobins widely distributed in bacteria, unicellular eukaryotes, and plants. J Biol Chem. 2001 Nov 5;277(2):871–874. doi: 10.1074/jbc.R100058200. [DOI] [PubMed] [Google Scholar]
  43. Wittwer A. J., Wagner C. Identification of the folate-binding proteins of rat liver mitochondria as dimethylglycine dehydrogenase and sarcosine dehydrogenase. Flavoprotein nature and enzymatic properties of the purified proteins. J Biol Chem. 1981 Apr 25;256(8):4109–4115. [PubMed] [Google Scholar]
  44. Wong P. S., Hyun J., Fukuto J. M., Shirota F. N., DeMaster E. G., Shoeman D. W., Nagasawa H. T. Reaction between S-nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry. Biochemistry. 1998 Apr 21;37(16):5362–5371. doi: 10.1021/bi973153g. [DOI] [PubMed] [Google Scholar]
  45. Zeng H., Spencer N. Y., Hogg N. Metabolism of S-nitrosoglutathione by endothelial cells. Am J Physiol Heart Circ Physiol. 2001 Jul;281(1):H432–H439. doi: 10.1152/ajpheart.2001.281.1.H432. [DOI] [PubMed] [Google Scholar]
  46. van Ophem P. W., Van Beeumen J., Duine J. A. NAD-linked, factor-dependent formaldehyde dehydrogenase or trimeric, zinc-containing, long-chain alcohol dehydrogenase from Amycolatopsis methanolica. Eur J Biochem. 1992 Jun 1;206(2):511–518. doi: 10.1111/j.1432-1033.1992.tb16954.x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES