Mycothiol disulfide reductase: solid phase synthesis and evaluation of alternative substrate analogues

Stewart, Matthew J; Jothivasan, Vishnu Karthik; Rowan, Andrew S.; Wagg, Jennifer; Hamilton, Chris J.

doi:10.1039/b716380k

Cited by 20 publications

(15 citation statements)

References 27 publications

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“…Mycothiol [1-D- myo -inosityl-2-( N -acetylcysteinyl)amido-2-deoxy-α-D-glucopyranoside], an abundant low molecular weight thiol found at millimolar concentrations in most actinomycetes, serves as the major thiol redox buffer for the cell (69). In the presence of methyl-mycothiol (31), a synthetic analogue of mycothiol, and thioredoxin, the extent of cluster loss was significantly reduced compared to an untreated sample. Furthermore, methyl-mycothiol also significantly reduced the sensitivity of native WhiD to low pH, with only a ~3-fold increase in the rate constant upon decreasing the pH from 7.0 to 4.5.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of [4Fe-4S]-Containing and Cluster-Free Forms of Streptomyces WhiD

et al. 2009

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WhiD, a member of the WhiB-like (Wbl) family of iron-sulfur proteins found exclusively within the actinomycetes, is required for the late stages of sporulation in Streptomyces coelicolor. Like all other Wbl proteins, WhiD has not so far been purified in a soluble form that contains a significant amount of cluster and characterization has relied on cluster-reconstituted protein. Thus, a major goal in Wbl research is to obtain and characterize native protein containing iron-sulfur clusters. Here we report the analysis of S. coelicolor WhiD purified anaerobically from E. coli as a soluble protein containing a single [4Fe-4S] 2+ cluster ligated by four cysteines. Upon exposure to oxygen, spectral features associated with the [4Fe-4S] cluster were lost in a slow reaction that unusually yielded apo-WhiD directly without significant concentrations of cluster intermediates. This process was found to be highly pH dependent with an optimal stability observed between pH 7.0 and 8.0. Low molecular weight thiols, including a mycothiol analogue and thioredoxin, exerted a small but significant protective effect against WhiD cluster loss, an activity that could be of physiological importance.[4Fe-4S] 2+ WhiD was found to react much more rapidly with superoxide than with either oxygen or hydrogen peroxide, which may also be of physiological significance. Loss of the [4Fe-4S] cluster to form apo-protein destabilized the protein fold significantly, but did not lead to complete unfolding. Finally, apo-WhiD exhibited negligible activity in an insulin-based disulfide reductase assay demonstrating that it does not function as a general protein disulfide reductase.WhiB-like (Wbl) proteins are found exclusively in the actinomycetes, a phylum of Grampositive bacteria that includes Streptomyces, the most abundant source of clinically important antibiotics and other bioactive molecules, and medically important pathogens such as Mycobacterium tuberculosis and Corynebacterium diphtheriae (1,2). Disruption of wbl genes has shown that Wbl proteins play critical roles in the biology of both Streptomyces and Mycobacterium (1,3). In Streptomyces coelicolor, WhiB, the founding member of the Wbl 1To whom correspondence should be addressed: A. J. Thomson Table S1; The reactivity of the [4Fe-4S] cluster monitored by CD spectroscopy is shown in Figure S1; The reactivity of the [4Fe-4S] cluster with superoxide is shown in Figure S2; Analysis of apo-and as isolated WhiD by SDS-PAGE is shown in Figure S3; EPR spectroscopy data following oxidation of WhiD is shown in Figure S4. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 December 29. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript family, is required for the initiation of sporulation septation, and WhiD, the subject of this report, is required for the late stages of sporulation (1,3,4). In M. tuberculosis, Wbl proteins have been implicated in...

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Section: Discussionmentioning

confidence: 99%

“…Low molecular weight thiols were obtained from Sigma Aldrich and were typically ≥97% purity. Methyl mycothiol was a kind gift (Dr Chris Hamilton, UEA); see Stewart et al (31) for further details. The pH dependence of cluster stability was also investigated using assay buffer containing 2 mM GSH at varying pH values.…”

Section: Methodsmentioning

confidence: 99%

Characterization of [4Fe-4S]-Containing and Cluster-Free Forms of Streptomyces WhiD

et al. 2009

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“…26 In a previous study of the substrate specificity of mtr the suitability of a few different aglycons in lieu of inositol in mycothiol analogs was explored. 27 The benzyl group was found to be the most suitable. This finding was rationalized on the basis that inositol has a hydrophobic 'patch' on the surface of the six-membered ring which displays three axial hydrogens.…”

Section: Synthesis Of Mycothiol Analogs In Which Naphthoquinolyl Carbmentioning

confidence: 99%

Conjugates of plumbagin and phenyl-2-amino-1-thioglucoside inhibit MshB, a deacetylase involved in the biosynthesis of mycothiol

Gammon

Steenkamp

Mavumengwana

et al. 2010

Bioorganic & Medicinal Chemistry

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“…For residues attached to the carboxyl group of cysteine, no activity is seen without the GlcN residue. The Ins residue can be replaced by a benzyl residue with only a minor loss of activity, and this derivative represents a synthetically accessible alternative substrate potentially useful in screening for Mtr inhibitors (137). All substitutions at the amino residue of cysteine, including simple removal of the acetyl moiety or its replacement by a formyl residue, produce significant losses of activity.…”

Section: Mtr the Msh Disulfide Reductase (Mycothione Reductase)mentioning

confidence: 99%

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

Newton

Buchmeier

Fahey

2008

Microbiol Mol Biol Rev

310

293

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SUMMARY 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 σB and σR 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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Mycothiol disulfide reductase: solid phase synthesis and evaluation of alternative substrate analogues

Cited by 20 publications

References 27 publications

Characterization of [4Fe-4S]-Containing and Cluster-Free Forms of Streptomyces WhiD

Characterization of [4Fe-4S]-Containing and Cluster-Free Forms of Streptomyces WhiD

Conjugates of plumbagin and phenyl-2-amino-1-thioglucoside inhibit MshB, a deacetylase involved in the biosynthesis of mycothiol

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

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