title: A mycothiol synthase mutant of Mycobacterium smegmatis produces novel thiols and has an altered thiol redox status.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0314, USA.
authors.forename: Gerald L
authors.initials: GL
authors.lastname: Newton
authors.forename: Philong
authors.initials: P
authors.lastname: Ta
authors.forename: Robert C
authors.initials: RC
authors.lastname: Fahey
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism; D001555:Benzene Derivatives / Q000633:toxicity; D003545:Cysteine; D004251:DNA Transposable Elements; D004187:Disaccharides / Q000096:biosynthesis*; D006020:Glycopeptides; D006861:Hydrogen Peroxide / Q000633:toxicity; D007294:Inositol; D016254:Mutagenesis, Insertional; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics*; D016877:Oxidants / Q000633:toxicity; D010084:Oxidation-Reduction; D011720:Pyrazoles; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000633:toxicity
chemical_list: D000214:Acyl Coenzyme A; D001555:Benzene Derivatives; D004251:DNA Transposable Elements; D004187:Disaccharides; D006020:Glycopeptides; D016877:Oxidants; D011720:Pyrazoles; D013438:Sulfhydryl Compounds; C089265:mycothiol; D007294:Inositol; D000105:Acetyl Coenzyme A; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; C012046:succinyl-coenzyme A; D000123:Acetyltransferases; C470312:acetyl-CoA 1-D-myo-inosityl 2-L-cysteinylamido-2-deoxy-alpha-D-glucopyranoside acetyltransferase; D003545:Cysteine; C007164:cumene hydroperoxide