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.

FIG. 1.

Reaction catalyzed by mycothiol synthase (MshD) and the structures for novel thiols produced by the mycothiol synthase mutant mshD::Tn5.

FIG. 2.

Cellular thiol composition during growth of M. smegmatis mc²155 (A) and M. smegmatis mshD::Tn5 (B). •, A₆₀₀; ▾, MSH; ▴, CoA; ♦, Cys; ○, fCys-GlcN-Ins; ▪, Cys-GlcN-Ins; □, succ-Cys-GlcN-Ins. Results from two independent cultures are shown; average deviation from mean was ≤20%, except for Cys-GlcN-Ins (where the average deviation was 30%).

FIG. 3.

Cellular redox status during growth of M. smegmatis mc²155 (A) and M. smegmatis mshD::Tn5 (B). •, A₆₀₀; ▾, MSH; ♦, Cys; ○, fCys-GlcN-Ins; □, succ-Cys-GlcN-Ins; ▪, Cys-GlcN-Ins. Results from two independent cultures are shown. The average deviation from the mean was ≤30%, except for the results shown in panel B, where the average deviation was 45% for MSH and 55% for succ-Cys-GlcN-Ins.

FIG. 4.

Chemical synthesis of acyl-Cys-GlcN-Ins from a reaction of 250 μM Cys-GlcN-Ins with 1 mM of propionyl-CoA to produce prop-Cys-GlcN-Ins (○), of acetyl-CoA to produce MSH (▿) and of succinyl-CoA to produce succ-Cys-GlcN-Ins (□) in 50 mM HEPES, pH 7.4, containing 1 mM DTT at 30°C. Lines correspond to the second-order rates calculated from the rate constants given in the text. The data shown are representative of three independent experiments which produced similar results.

FIG. 5.

Autoxidation of thiols in the presence of 50 mM HEPES, pH 7.3, containing 0.3 μM CuSO₄. (A) ○, 200 μM MSH; •, 200 μM Cys-GlcN-Ins. (B) 100 μM MSH (○) plus 100 μM Cys-GlcN-Ins (•); total thiol (□). Three independent experiments all produced results essentially identical to the one shown.

FIG. 6.

Reduction of 100 μM succ-Cys-GlcN-Ins disulfide preincubated for 30 min with 30 μM of the thiol fCys-GlcN-Ins (•) or MSH (▾) by Mtr as monitored by NADPH absorption at 340 nm.