Preparation and utilization of a reagent for the isolation and purification of low-molecular-mass thiols

Steenkamp, Daniël J.; Vogt, Ryan N

doi:10.1016/j.ab.2003.10.011

Cited by 13 publications

(13 citation statements)

References 20 publications

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“…A limited range of 2,4-naphthoquinones, tethered via spacers of varying length to the amino group of 1-S-thiophenyl-2-deoxy-2-aminoglucopyranose, were synthesized as potential inhibitors of MshB using a strategy similar to that reported by Salmon-Chemin et al 20 Some of these compounds were amongst the most potent inhibitors of MshB yet found. The substrate specificity of Mca was found to overlap not only with that of MshB, as previously reported, but also with that of the disulfide reductase, mtr, since both mtr and Mca utilized the heterodisulfide 2-S-mycothiolyl-6-hydroxynaphthyldisulfide 21 …”

Section: Introductionsupporting

confidence: 55%

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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Section: Introductionsupporting

confidence: 55%

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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“…MSH was isolated from M. smegmatis as described previously [25]. The E. coli-mycobacteria shuttle vector pSD26 was a gift from Dr W. R. Jacobs (Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, U.S.A.).…”

Section: Methodsmentioning

confidence: 99%

The metabolism of nitrosothiols in the mycobacteria: identification and characterization of S-nitrosomycothiol reductase

Vogt

Steenkamp

Zheng

et al. 2003

Biochemical Journal

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When grown in culture Mycobacterium smegmatis metabolized S-nitrosoglutathione to oxidized glutathione and nitrate, which suggested a possible involvement of an S-nitrosothiol reductase and mycobacterial haemoglobin. The mycothiol-dependent formaldehyde dehydrogenase from M. smegmatis was purified by a combination of Ni2+-IMAC (immobilized metal ion affinity chromatography), hydrophobic interaction, anion-exchange and affinity chromatography. The enzyme had a subunit molecular mass of 38263 kDa. Steady-state kinetic studies indicated that the enzyme catalyses the NAD+-dependent conversion of S-hydroxymethylmycothiol into formic acid and mycothiol by a rapid-equilibrium ordered mechanism. The enzyme also catalysed an NADH-dependent decomposition of S-nitrosomycothiol (MSNO) by a sequential mechanism and with an equimolar stoichiometry of NADH:MSNO, which indicated that the enzyme reduces the nitroso group to the oxidation level of nitroxyl. Vmax for the MSNO reductase reaction indicated a turnover per subunit of approx. 116700 min(-1), which was 76-fold faster than the formaldehyde dehydrogenase activity. A gene, Rv2259, annotated as a class III alcohol dehydrogenase in the Mycobacterium tuberculosis genome was cloned and expressed in M. smegmatis as the C-terminally His6-tagged product. The purified recombinant enzyme from M. tuberculosis also catalysed both activities. M. smegmatis S-nitrosomycothiol reductase converted MSNO into the N -hydroxysulphenamide, which readily rearranged to mycothiolsulphinamide. In the presence of MSNO reductase, M. tuberculosis HbN (haemoglobin N) was converted with low efficiency into metHbN [HbN(Fe3+)] and this conversion was dependent on turnover of MSNO reductase. These observations suggest a possible route in vivo for the dissimilation of S-nitrosoglutathione.

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“…Intracellular ERG was extracted by using a previously described method, with modifications (26,27). Liquid M. smegmatis cultures were grown to the specified optical density at 600 nm (OD 600 ), harvested by centrifugation, and resuspended in lysis buffer (40% acetonitrile, 0.25 M perchloric acid).…”

Section: Methodsmentioning

confidence: 99%

Ergothioneine Is a Secreted Antioxidant in Mycobacterium smegmatis

Emani

Williams

Wiid

et al. 2013

Antimicrob Agents Chemother

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c Ergothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle of Mycobacterium smegmatis revealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant of M. smegmatis lacking egtD (MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion of egtD from wild-type M. smegmatis and an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG-and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protecting M. smegmatis against oxidative stress and that ERG is able to partly compensate for the loss of MSH. G lutathione (GSH) is a thiol known for its efficient detoxification of reactive oxygen species, reactive nitrogen species, and free radicals in eukaryotes. Mycobacteria do not synthesize GSH but produce two low-molecular-weight thiols,and ergothioneine (2-mercaptohistidine trimethylbetaine) (ERG) (4, 5). Four genes are involved in MSH biosynthesis in mycobacteria, namely, mshA, mshB, mshC, and mshD, and mutants harboring deletions in mshB, mshC, and mshD produce different levels of MSH due to the ability of other enzymes to partially compensate for their loss (6, 7). MSH-deficient mutants of Mycobacterium smegmatis show increased sensitivity to oxidative stress, alkylating agents, and a range of antibiotics, including erythromycin, azithromycin, vancomycin, penicillin G, streptomycin, and rifampin, but exhibit increased resistance to isoniazid (INH) and ethionamide (ETH) (8, 9). The MSH-deficient ⌬mshA mutant of Mycobacterium tuberculosis requires catalase during in vitro growth, implicating MSH in detoxifying reactive oxygen species (10).ERG biosynthetic genes (egtA, egtB, egtC, egtD, and egtE) were recently identified in M. smegmatis (11). Although several lines of evidence support the cytoprotective and antioxidative role of ERG in eukaryotes (12), bacteria (13), and, recently, fungi (14), nothing is known of its role in mycobacteria. ERG has also been implicated in modulating the immune response (15) and in the inhibition of metalloenzymes, preventing the copper-induced oxidation of DNA and protein due to its metal-chelating properties (16,17). Eukaryotes obtain ERG from their diet, and its accumulation in cells is dependent on the activity of a highly specific transporter, OCTN1, since the zwitterionic nature of ERG prevents it from crossing the plasma membrane (18,19). In an M. smegmatis ⌬mshA mutant, which is MSH deficient, the levels of ERG and the organic hydroperoxide resistance (Ohr) protein are elevated, suggesting that ERG may partly compensate for the loss of MSH (20). This may exp...

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Preparation and utilization of a reagent for the isolation and purification of low-molecular-mass thiols

Cited by 13 publications

References 20 publications

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

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

The metabolism of nitrosothiols in the mycobacteria: identification and characterization of S-nitrosomycothiol reductase

Ergothioneine Is a Secreted Antioxidant in Mycobacterium smegmatis

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