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*CUMENE HYDROPEROXIDE
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Journal of Bacteriology, November 2005, p. 7309-7316, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7309-7316.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Mycothiol Synthase Mutant of Mycobacterium smegmatis Produces Novel Thiols and Has an Altered Thiol Redox Status

Gerald L. Newton, Philong Ta, and Robert C. Fahey*

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California

Received 8 June 2005/ Accepted 8 August 2005

Mycobacteria and other actinomycetes do not produce glutathione but make mycothiol (MSH; AcCys-GlcN-Ins) that has functions similar to those of glutathione and is essential for growth of Mycobacterium tuberculosis. Mycothiol synthase (MshD) catalyzes N acetylation of Cys-GlcN-Ins to produce MSH in Mycobacterium smegmatis mc2155, and Cys-GlcN-Ins is maintained at a low level. The mycothiol synthase mutant, the mshD::Tn5 mutant, produces high levels of Cys-GlcN-Ins along with two novel thiols, N-formyl-Cys-GlcN-Ins and N-succinyl-Cys-GlcN-Ins, and a small amount of MSH. The nonenzymatic reaction of acyl-coenzyme A (CoA) with Cys-GlcN-Ins to produce acyl-Cys-GlcN-Ins is a facile reaction under physiologic conditions, with succinyl-CoA being an order of magnitude more reactive than acetyl-CoA. The uncatalyzed reaction rates are adequate to account for the observed production of N-succinyl-Cys-GlcN-Ins and MSH under physiologic conditions. It was shown that the N-acyl-Cys-GlcN-Ins compounds are maintained in a substantially reduced state in the mutant but that Cys-GlcN-Ins exists in disulfide forms at 5 to 40% at different stages of growth. MSH was able to facilitate reduction of N-succinyl-Cys-GlcN-Ins disulfide through thiol-disulfide exchange, but N-formyl-Cys-GlcN-Ins was ineffective. The oxidized state of Cys-GlcN-Ins in cells appears to result from a high susceptibility to autoxidation and a low capacity of the cell to reduce its disulfide forms. The mutant exhibited no enhanced sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, or cumene hydroperoxide relative to the parent strain, suggesting that the most abundant thiol, N-formyl-Cys-GlcN-Ins, functions as a substitute for MSH.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0314. Phone: (858) 534-2163. Fax: (858) 534-4864. E-mail: rcfahey{at}ucsd.edu.

Journal of Bacteriology, November 2005, p. 7309-7316, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7309-7316.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Newton, G. L., Buchmeier, N., Fahey, R. C. (2008). Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria. Microbiol. Mol. Biol. Rev. 72: 471-494 [Abstract] [Full Text]  
  • Vetting, M. W., Frantom, P. A., Blanchard, J. S. (2008). Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum: SUBSTRATE-ASSISTED CATALYSIS. J. Biol. Chem. 283: 15834-15844 [Abstract] [Full Text]  
  • Bzymek, K. P., Newton, G. L., Ta, P., Fahey, R. C. (2007). Mycothiol Import by Mycobacterium smegmatis and Function as a Resource for Metabolic Precursors and Energy Production. J. Bacteriol. 189: 6796-6805 [Abstract] [Full Text]  
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