Mycothiol Import by Mycobacterium smegmatis and Function as a Resource for Metabolic Precursors and Energy Production

Abstract

Mycothiol ([MSH]AcCys-GlcN-Ins, where Ac is acetyl) is the major thiol produced by Mycobacterium smegmatis and other actinomycetes. Mutants deficient in MshA (strain 49) or MshC (transposon mutant Tn1) of MSH biosynthesis produce no MSH. However, when stationary phase cultures of these mutants were incubated in medium containing MSH, they actively transported it to generate cellular levels of MSH comparable to or greater than the normal content of the wild-type strain. When these MSH-loaded mutants were transferred to MSH-free preconditioned medium, the cellular MSH was catabolized to generate GlcN-Ins and AcCys. The latter was rapidly converted to Cys by a high deacetylase activity assayed in extracts. The Cys could be converted to pyruvate by a cysteine desulfhydrase or used to regenerate MSH in cells with active MshC. Using MSH labeled with [U-14 C]cysteine or with [6-3 H]GlcN, it was shown that these residues are catabolized to generate radiolabeled products that are ultimately lost from the cell, indicating extensive catabolism via the glycolytic and Krebs cycle pathways. These findings, coupled with the fact the myo-inositol can serve as a sole carbon source for growth of M. smegmatis, indicate that MSH functions not only as a protective cofactor but also as a reservoir of readily available biosynthetic precursors and energy-generating metabolites potentially important under stress conditions. The half-life of MSH was determined in stationary phase cells to be ϳ50 h in strains with active MshC and 16 ؎ 3 h in the MshC-deficient mutant, suggesting that MSH biosynthesis may be a suitable target for drugs to treat dormant tuberculosis.

Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

“…Glucose was proposed to be metabolized through pyruvate and the Krebs cycle, with the 3 H label lost to the medium as water. AcCys, produced from MSH by Mca, was deacetylated by an active deacetylase detected in cell extracts (15). The resulting [U-…”

“…The unexpected discoveries that M. smegmatis mutants devoid of MSH are able to actively import MSH and to accumulate MSH to levels at or above the wild-type content opened the way to examine the turnover of MSH (15). When stationary-phase cells of the MshA-and MshC-deficient mutants were loaded with MSH and resuspended in conditioned stationaryphase medium and the MSH content followed with time, it was found that their half-lives for loss of MSH were ϳ50 h and ϳ16 h, respectively.…”

“…Glutathionylation of proteins, which is the production of protein-SSG disulfides, was recently explored as a regulatory process under both oxidative stress and constitutive conditions (20,31,34,49,67) and was earlier shown to be important in the dormancy of fungal spores (10,26). Analogous mycothiolation of proteins could also be important, and there is some indication that mycothiolation of proteins occurs in mycobacteria (15).…”

“…14 C]cysteine was converted to pyruvate by Cys desulfhydrase (15,156), recently identified as Rv3025c in M. tuberculosis (131). Pyruvate was metabolized through the Krebs cycle, with the loss of label as carbon dioxide.…”

Thiol-Dependent Peroxidases in Mycobacterium tuberculosis Antioxidant Defense

“…It participates in drug detoxification pathways by forming adducts with alkylating agents and antibiotics that are subsequently cleaved by MSH S-conjugate amidase to generate a mercapturic acid (excreted outside the cell) and glucosamine inositol (used to regenerate MSH) (Newton et al, 2000). MSH can function as a resource for metabolic precursors and for energy production (Bzymek et al, 2007). Mycothiol-deficient M. smegmatis strains are more sensitive to • NO-and H 2 O 2 -mediated toxicity than wild type strains (Rawat et al, 2002;Miller et al, 2007).…”