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Review
. 2008 Sep;72(3):471-94.
doi: 10.1128/MMBR.00008-08.

Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria

Gerald L Newton  1 , Nancy Buchmeier, Robert C Fahey
Affiliations
Review

Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria

Gerald L Newton et al. Microbiol Mol Biol Rev. 2008 Sep.

Abstract

Mycothiol (MSH; AcCys-GlcN-Ins) is the major thiol found in Actinobacteria and has many of the functions of glutathione, which is the dominant thiol in other bacteria and eukaryotes but is absent in Actinobacteria. MSH functions as a protected reserve of cysteine and in the detoxification of alkylating agents, reactive oxygen and nitrogen species, and antibiotics. MSH also acts as a thiol buffer which is important in maintaining the highly reducing environment within the cell and protecting against disulfide stress. The pathway of MSH biosynthesis involves production of GlcNAc-Ins-P by MSH glycosyltransferase (MshA), dephosphorylation by the MSH phosphatase MshA2 (not yet identified), deacetylation by MshB to produce GlcN-Ins, linkage to Cys by the MSH ligase MshC, and acetylation by MSH synthase (MshD), yielding MSH. Studies of MSH mutants have shown that the MSH glycosyltransferase MshA and the MSH ligase MshC are required for MSH production, whereas mutants in the MSH deacetylase MshB and the acetyltransferase (MSH synthase) MshD produce some MSH and/or a closely related thiol. Current evidence indicates that MSH biosynthesis is controlled by transcriptional regulation mediated by sigma(B) and sigma(R) in Streptomyces coelicolor. Identified enzymes of MSH metabolism include mycothione reductase (disulfide reductase; Mtr), the S-nitrosomycothiol reductase MscR, the MSH S-conjugate amidase Mca, and an MSH-dependent maleylpyruvate isomerase. Mca cleaves MSH S-conjugates to generate mercapturic acids (AcCySR), excreted from the cell, and GlcN-Ins, used for resynthesis of MSH. The phenotypes of MSH-deficient mutants indicate the occurrence of one or more MSH-dependent S-transferases, peroxidases, and mycoredoxins, which are important targets for future studies. Current evidence suggests that several MSH biosynthetic and metabolic enzymes are potential targets for drugs against tuberculosis. The functions of MSH in antibiotic-producing streptomycetes and in bioremediation are areas for future study.

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Figures

FIG. 1.
FIG. 1.
(A) Structure of MSH. (B) Conformation of mycothiol-S-bimane determined by NMR. (Reprinted in part with permission from reference . Copyright 2003 American Chemical Society.) (C) Cysteine complexed to heavy metal. (D) Transition state for S-N-acyl migration of S-acylcysteine thioesters.
FIG. 2.
FIG. 2.
Biosynthesis of MSH. myo-Inositol-1-phosphate synthase (Ino1) generates Ins-P, MSH glycosyltransferase (MshA) links Ins-P to GlcNAc, MSH phosphatase (MshA2) generates GlcNAc-Ins, MSH deacetylase (MshB) produces GlcN-Ins, MSH ligase (MshC) links Cys with GlcN-Ins, and MSH synthase (MSH acetyltransferase; MshD) acetylates Cys-GlcN-Ins to produce MSH. Note that myo-inositol has a plane of symmetry through C-2 and C-5, making C-1 and C-3 equivalent so GlcN-Ins-P can be named as a derivative of 1l-Ins-1-P or 1d-Ins-3-P, and that both systems are used.
FIG. 3.
FIG. 3.
(A) PyMOL structure of selected active-site residues of MshA from Corynebacterium glutamicum in complex with UDP and Ins-P (146). (B) Summary of proposed catalytic mechanism for production of GlcNAc-Ins-P by MshA (146).
FIG. 4.
FIG. 4.
Proposed mechanism for the M. tuberculosis deacetylase MshB (71). The active site is formed with Asp15, His13, and His147 coodinating a Zn ion required for catalysis and protein stability. The active-site Zn also polarizes the acetyl carbon-oxygen bond of bound GlcNAc-Ins for attack by a hydroxyl ion generated from water by protonation of Asp15. The forming acetate is hydrogen bonded to His144 or Asp15 to complete the hydrolysis.
FIG. 5.
FIG. 5.
Relative rates of M. tuberculosis MSH deacetylase (MshB) cleavage of amide bonds in acyl glucosamine derivatives. The substrate specificity of MshB overlaps that of MSH S-conjugate amidase (Mca) (see Fig. 11). The natural substrate of MshB is GlcNAc-Ins, whose rate is set at 100 as a reference.
FIG. 6.
FIG. 6.
(A) Bi uni uni bi ping pong kinetic mechanism for M. smegmatis MshC (28). (B) Structures of the Cys adenylate intermediate and its Cys sulfamoyl adenylate inhibitor analog.
FIG. 7.
FIG. 7.
Proposed catalytic mechanism for M. tuberculosis MshD (148). The acetyl group from acetyl-CoA is transferred to the Cys amine of Cys-GlcN-Ins in a bound ternary complex, with subsequent release of CoASH, in a mechanism similar to that of other GCN5 acetyltransferases.
FIG. 8.
FIG. 8.
Summary of well-characterized MSH-dependent reactions where MSH is either a substrate or cofactor. MSH is the redox buffer for Actinobacteria, is oxidized by cellular oxidants, [O], and is maintained in a highly reduced status by MSH disulfide reductase (Mtr). Formaldehyde (HCOOH) and MSNO are converted to formate and MSH sulfinamide (MSONH2), respectively, by the alcohol dehydrogenase (AdhE2)/MSNO reductase (MscR). Thiol-reactive drugs form MSR that are cleaved by MSH S-conjugate amidase (Mca) to produce the N-acetyl-CyS-conjugate (AcCySR), which is excreted. The pseudodisaccharide GlcN-Ins is retained in the cell and reenters the MSH biosynthesis pathway. Another important function of Mca is the degradation of MSH to provide cysteine (from acetylcysteine [AcCys]) and GlcN-Ins as needed for other metabolic pathways. MSH is also required for the metabolism of 3-hydroxybenzoate; the MSH-dependent step is the isomerization of maleyl-pyruvate to fumaryl-pyruvate by maleylpyruvate isomerase (see Fig. 16 for more details).
FIG. 9.
FIG. 9.
Proposed catalytic mechanism for reduction of MSSM by MSSM reductase, Mtr (110). The oxidized enzyme (Mtrox) binds NADPH, and the active-site disulfide (C-44-S-S-C-39) is reduced by electrons from NADPH mediated by flavin adenine dinucleotide to generate reduced enzyme (Mtrred). Mtrred binds the substrate disulfide, MSSM, and releases the oxidized pyridine nucleotide, NADP+. Cleavage of bound MSSM by active-site C-39S⊖ to generate bound M-S-S-C-39 is followed by cleavage of M-S-S-C-39 by C-44S⊖ and release of two MSH molecules to regenerate Mtrox. ⊖, negative charge.
FIG. 10.
FIG. 10.
MSH-dependent detoxification of thiol-reactive drugs and metabolites, as reported for M. smegmatis and S. coelicolor (86, 106, 136). The thiol-reactive reagent mBBr reacts with MSH, and the MSR is hydrolyzed by MSH S-conjugate amidase (Mca) to GlcN-Ins and the excreted mercapturic acid AcCySmB. GlcN-Ins is retained in the cell and recycled for MSH synthesis, using MshC and MshD.
FIG. 11.
FIG. 11.
Substrate specificity of M. tuberculosis MSH S-conjugate amidase (Mca) for MSR (136), where the MSH moiety is held constant and the S-conjugate moiety (R1) is varied (A) or the S-conjugate moiety (bimane) is held constant and the MSH moiety is varied, from Cys-GlcN-Ins to formyl Cys-GlcN-Ins to MSH (B).
FIG. 12.
FIG. 12.
Antibiotics produced in Actinobacteria fermentation broths from Streptomyces violaceruber Tü (granaticin A and its mercapturic acid, granaticin MA) (A) and Streptomyces sp. strain E/784 (naphthomycin A and its mercapturic acid, naphthomycin G) (B).
FIG. 13.
FIG. 13.
Human 20S proteasome inhibitors produced by Streptomyces JS360 cinnabaramide A and its mercapturic acid, cinnabaramide F (134).
FIG. 14.
FIG. 14.
Hydroxamate inhibitors of MSH S-conjugate amidase. H, a bromotyrosine alkyloid from the marine sponge Oceanapia sp. (100), and the closely related synthetic analog EXEG1706 (113) are shown.
FIG. 15.
FIG. 15.
Two known activities of the alcohol dehydrogenase previously annotated AdhE2 in the M. tuberculosis genome. (A) MSH-dependent formaldehyde dehydrogenase reaction with MscR, generating formate in Amycolatopsis methanolica and Rhodococcus erythropolis (80), and with M. smegmatis MscR (149). (B) MSNO reaction with MscR, generating MSH sulfinic acid (MSO2H) in M. smegmatis (149).
FIG. 16.
FIG. 16.
Thiol-dependent maleylpyruvate isomerase function in the gentisate pathway.
FIG. 17.
FIG. 17.
Fate of [U-14C]cysteine-labeled (red) and [6-3H]glucosamine-labeled (green) MSH loaded for 3 h into stationary-phase cells of MshC-deficient M. smegmatis, resuspended after being washed in conditioned stationary-phase medium, and incubated for 24 h with sampling. Data presented represent the percent distribution of label after 24 h (15). Pellet, amount of label in resuspended pellet after extraction in hot 50% acetonitrile; void, amount of label eluted in void volume of mBBr-labeled extract separated by reversed-phase C18 HPLC (nonthiol glycolysis and Krebs cycle intermediates); medium, total amount of label in medium; lost, amount of label not recovered in cells or medium.
FIG. 18.
FIG. 18.
The cyclohexyl thioglycoside substrate analog of MSmB (R1) is an efficient substrate of Mca, and the nonsubstrate analog (R2) is an inhibitor of Mca and MshB.
FIG. 19.
FIG. 19.
Structure of MshC inhibitor NTF1836.
FIG. 20.
FIG. 20.
Quinone-subversive substrates of M. tuberculosis MSSM reductase Mtr (70).
FIG. 21.
FIG. 21.
Synthetic nonreacting analogs of the substrate (GlcNAc-Ins) for the MSH deacetylase MshB in which the NH residue is replaced by CH2 (G2) and the corresponding alcohol (G1); both G1 and G2 inhibit MSH biosynthesis in M. smegmatis, about twofold, at 200 μg/ml (33).
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