Archives of Microbiology

, Volume 190, Issue 5, pp 547–557

An N-acyl homolog of mycothiol is produced in marine actinomycetes

  • Gerald L. Newton
  • Paul R. Jensen
  • John B. MacMillan
  • William Fenical
  • Robert C. Fahey
Original Paper


Marine actinomycetes have generated much recent interest as a potentially valuable source of novel antibiotics. Like terrestrial actinomycetes the marine actinomycetes are shown here to produce mycothiol as their protective thiol. However, a novel thiol, U25, was produced by MAR2 strain CNQ703 upon progression into stationary phase when secondary metabolite production occurred and became the dominant thiol. MSH and U25 were maintained in a reduced state during early stationary phase, but become significantly oxidized after 10 days in culture. Isolation and structural analysis of the monobromobimane derivative identified U25 as a homolog of mycothiol in which the acetyl group attached to the nitrogen of cysteine is replaced by a propionyl residue. This N-propionyl-desacetyl-mycothiol was present in 13 of the 17 strains of marine actinomycetes examined, including five strains of Salinispora and representatives of the MAR2, MAR3, MAR4 and MAR6 groups. Mycothiol and its precursor, the pseudodisaccharide 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol, were found in all strains. High levels of mycothiol S-conjugate amidase activity, a key enzyme in mycothiol-dependent detoxification, were found in most strains. The results demonstrate that major thiol/disulfide changes accompany secondary metabolite production and suggest that mycothiol-dependent detoxification is important at this developmental stage.


Mycothiol N-propionyl-desacetyl-mycothiol Marine actinomycetes Salinispora arencola Mca 


  1. Anderberg SJ, Newton GL, Fahey RC (1998) Mycothiol biosynthesis and metabolism: cellular levels of potential intermediates in the biosynthesis and degradation of mycothiol. J Biol Chem 273:30391–30397PubMedCrossRefGoogle Scholar
  2. Boshoff HI, Barry CE 3rd (2005) Tuberculosis—metabolism and respiration in the absence of growth. Nat Rev Microbiol 3:70–80PubMedCrossRefGoogle Scholar
  3. Brock M (2005) Generation and phenotypic characterization of Aspergillus nidulans methylisocitrate lyase deletion mutants: methylisocitrate inhibits growth and conidiation. Appl Environ Microbiol 71:5465–5475PubMedCrossRefGoogle Scholar
  4. Brock M, Buckel W (2004) On the mechanism of action of the antifungal agent propionate. Eur J Biochem 271:3227–3241PubMedCrossRefGoogle Scholar
  5. Buchmeier N, Fahey RC (2006) The mshA gene encoding the glycosyltransferase of mycothiol biosynthesis is essential in Mycobacterium tuberculosis Erdman. FEMS Microbiol Lett 264:74–79PubMedCrossRefGoogle Scholar
  6. Buchmeier NA, Newton GL, Koledin T, Fahey RC (2003) Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. Mol Microbiol 47:1723–1732PubMedCrossRefGoogle Scholar
  7. Buchmeier NA, Newton GL, Fahey RC (2006) A mycothiol synthase mutant of Mycobacterium tuberculosis has an altered thiol-disulfide content and limited tolerance to stress. J Bacteriol 188:6245–6252PubMedCrossRefGoogle Scholar
  8. Bull AT, Stach JE (2007) Marine actinobacteria: new opportunities for natural product search and discovery. Trends Microbiol 15:491–499PubMedCrossRefGoogle Scholar
  9. Bull AT, Stach JE, Ward AC, Goodfellow M (2005) Marine actinobacteria: perspectives, challenges, future directions. Antonie Van Leeuwenhoek 87:65–79CrossRefGoogle Scholar
  10. Bzymek KP, Newton GL, Ta P, Fahey RC (2007) Mycothiol import by Mycobacterium smegmatis and function as a resource for metabolic precursors and energy production. J Bacteriol 189:6796–6805PubMedCrossRefGoogle Scholar
  11. Dalle-Donne I, Rossi R, Giustarini D, Colombo R, Milzani A (2007) S-glutathionylation in protein redox regulation. Free Radic Biol Med 43:883–898PubMedCrossRefGoogle Scholar
  12. Fahey RC, Newton GL (1987) Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography. Methods Enzymol 143:85–96PubMedCrossRefGoogle Scholar
  13. Fahey RC, Sundquist AR (1991) Evolution of glutathione metabolism. Adv Enzymol Relat Areas Mol Biol 64:1–53PubMedCrossRefGoogle Scholar
  14. Fahey RC, Brown WC, Adams WB, Worsham MB (1978) Occurrence of glutathione in bacteria. J Bacteriol 133:1126–1129PubMedGoogle Scholar
  15. Fenical W, Jensen PR (2006) Developing a new resource for drug discovery: marine actinomycete bacteria. Nat Chem Biol 2:666–673PubMedCrossRefGoogle Scholar
  16. Feling RH, Buchanan GO, Mincer TJ, Kauffman CA, Jensen PR, Fenical W (2003) Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus Salinospora. Angew Chem Int Ed Engl 42:355–357PubMedCrossRefGoogle Scholar
  17. Fiedler HP, Bruntner C, Bull AT, Ward AC, Goodfellow M, Potterat O, Puder C, Mihm G (2005) Marine actinomycetes as a source of novel secondary metabolites. Antonie Van Leeuwenhoek 87:37–42PubMedCrossRefGoogle Scholar
  18. Ghezzi P (2005) Regulation of protein function by glutathionylation. Free Radic Res 39:573–580PubMedCrossRefGoogle Scholar
  19. Hand CE, Honek JF (2005) Biological chemistry of naturally occurring thiols of microbial and marine origin. J Nat Prod 68:293–308PubMedCrossRefGoogle Scholar
  20. Hurd TR, Costa NJ, Dahm CC, Beer SM, Brown SE, Filipovska A, Murphy MP (2005) Glutathionylation of mitochondrial proteins. Antioxid Redox Signal 7:999–1010PubMedCrossRefGoogle Scholar
  21. Jardine MA, Spies HS, Nkambule CM, Gammon DW, Steenkamp DJ (2002) Synthesis of mycothiol, 1D-1-O-(2-[N-acetyl-L-cysteinyl]amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol, principal low molecular mass thiol in the actinomycetes. Bioorg Med Chem 10:875–881PubMedCrossRefGoogle Scholar
  22. Jensen PR, Gontang E, Mafnas C, Mincer TJ, Fenical W (2005a) Culturable marine actinomycete diversity from tropical Pacific Ocean sediments. Environ Microbiol 7:1039–1048PubMedCrossRefGoogle Scholar
  23. Jensen PR, Mincer TJ, Williams PG, Fenical W (2005b) Marine actinomycete diversity and natural product discovery. Antonie Van Leeuwenhoek 87:43–48PubMedCrossRefGoogle Scholar
  24. Jensen PR, Williams PG, Oh DC, Zeigler L, Fenical W (2007) Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora. Appl Environ Microbiol 73:1146–1152PubMedCrossRefGoogle Scholar
  25. Koburger T, Weibezahn J, Bernhardt J, Homuth G, Hecker M (2005) Genome-wide mRNA profiling in glucose starved Bacillus subtilis cells. Mol Genet Genomics 274:1–12PubMedCrossRefGoogle Scholar
  26. Koledin T, Newton GL, Fahey RC (2002) Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes. Arch Microbiol 178:331–337PubMedCrossRefGoogle Scholar
  27. Kwon HC, Kauffman CA, Jensen PR, Fenical W (2006) Marinomycins A-D, antitumor-antibiotics of a new structure class from a marine actinomycete of the recently discovered genus “marinispora”. J Am Chem Soc 128:1622–1632PubMedCrossRefGoogle Scholar
  28. Lee S, Rosazza JP (2004) First total synthesis of mycothiol and mycothiol disulfide. Org Lett 6:365–368PubMedCrossRefGoogle Scholar
  29. Maldonado LA, Fenical W, Jensen PR, Kauffman CA, Mincer TJ, Ward AC, Bull AT, Goodfellow M (2005) Salinispora arenicola gen. nov., sp. nov. and Salinispora tropica sp. nov., obligate marine actinomycetes belonging to the family Micromonosporaceae. Int J Syst Evol Microbiol 55:1759–1766PubMedCrossRefGoogle Scholar
  30. Masip L, Veeravalli K, Georgiou G (2006) The many faces of glutathione in bacteria. Antioxid Redox Signal 8:753–762PubMedCrossRefGoogle Scholar
  31. Mincer TJ, Jensen PR, Kauffman CA, Fenical W (2002) Widespread and persistent populations of a major new marine actinomycete taxon in ocean sediments. Appl Environ Microbiol 68:5005–5011PubMedCrossRefGoogle Scholar
  32. Newton GL, Fahey RC (1995) Determination of biothiols by bromobimane labeling and high-performance liquid chromatography. Methods Enzymol 251:148–166PubMedCrossRefGoogle Scholar
  33. Newton GL, Fahey RC (2002) Mycothiol biochemistry. Arch Microbiol 178:388–394PubMedCrossRefGoogle Scholar
  34. Newton GL, Fahey RC, Cohen G, Aharonowitz Y (1993) Low molecular weight thiols in streptomycetes and their potential role as antioxidants. J Bacteriol 175:2734–2742PubMedGoogle Scholar
  35. Newton GL, Bewley CA, Dwyer TJ, Horn R, Aharonowitz Y, Cohen G, Davies J, Faulkner DJ, Fahey RC (1995) The structure of U17 isolated from Streptomyces clavuligerus and its properties as an antioxidant thiol. Eur J Biochem 230:821–825PubMedCrossRefGoogle Scholar
  36. Newton GL, Arnold K, Price MS, Sherrill C, delCardayré SB, Aharonowitz Y, Cohen G, Fahey RC, Davis C (1996) Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J Bacteriol 178:1990–1995PubMedGoogle Scholar
  37. Newton GL, Unson MD, Anderberg SJ, Aguilera JA, Oh NN, delCardayré SB, Davies J, Av-Gay Y, Fahey RC (1999) Characterization of a Mycobacterium smegmatis mutant defective in 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside and mycothiol biosynthesis. Biochem Biophys Res Commun 255:239–244PubMedCrossRefGoogle Scholar
  38. Newton GL, Av-Gay Y, Fahey RC (2000a) N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. J Bacteriol 182:6958–6963PubMedCrossRefGoogle Scholar
  39. Newton GL, Av-Gay Y, Fahey RC (2000b) A novel mycothiol-dependent detoxification pathway in mycobacteria involving mycothiol S-conjugate amidase. Biochemistry 39:10739–10746PubMedCrossRefGoogle Scholar
  40. Newton GL, Koledin T, Gorovitz B, Rawat M, Fahey RC, Av-Gay Y (2003) The glycosyltransferase gene encoding the enzyme catalyzing the first step of mycothiol biosynthesis (mshA). J Bacteriol 185:3476–3479PubMedCrossRefGoogle Scholar
  41. Newton GL, Ta P, Fahey RC (2005) A mycothiol synthase mutant of Mycobacterium smegmatis produces novel thiols and has an altered thiol redox status. J Bacteriol 187:7309–7316PubMedCrossRefGoogle Scholar
  42. Newton GL, Ta P, Bzymek K, Fahey RC (2006) Biochemistry of the initial steps of mycothiol biosynthesis. J Biol Chem 281:33910–33920PubMedCrossRefGoogle Scholar
  43. Nicholas GM, Kovac P, Bewley CA (2002) Total synthesis and proof of structure of mycothiol bimane. J Am Chem Soc 124:3492–3493PubMedCrossRefGoogle Scholar
  44. Pandey AK, Sassetti CM (2008) Mycobacterial persistence requires the utilization of host cholesterol. Proc Natl Acad Sci USA 105:4376–4380PubMedCrossRefGoogle Scholar
  45. Park J-H, Roe J-H (2008) Mycothiol regulates and is regulated by a thiol-specific anti-sigma factor RsrA and sigmaR in Streptomyces coelicolor. Mol Microbiol 68:861–870PubMedCrossRefGoogle Scholar
  46. Patel MP, Blanchard JS (2001) Mycobacterium tuberculosis mycothione reductase: pH dependence of the kinetic parameters and kinetic isotope effects. Biochemistry 40:3119–3126Google Scholar
  47. Rawat M, Newton GL, Ko M, Martinez GJ, Fahey RC, Av-Gay Y (2002) Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals and antibiotics. Antimicrob Agents Chemother 46:3348–3355PubMedCrossRefGoogle Scholar
  48. Rawat M, Uppal M, Newton G, Steffek M, Fahey RC, Av-Gay Y (2004) Targeted mutagenesis of the Mycobacterium smegmatis mca gene, encoding a mycothiol-dependent detoxification protein. J Bacteriol 186:6050–6058PubMedCrossRefGoogle Scholar
  49. Sareen D, Steffek M, Newton GL, Fahey RC (2002) ATP-dependent L-cysteine:1D-myo-inosityl 2-amino-2-deoxy-α-D-glucopyranoside ligase, mycothiol biosynthesis enzyme MshC, is related to class I cysteinyl-tRNA synthetases. Biochemistry 41:6885–6890PubMedCrossRefGoogle Scholar
  50. Sareen D, Newton GL, Fahey RC, Buchmeier NA (2003) Mycothiol is essential for growth of Mycobacterium tuberculosis Erdman. J Bacteriol 185:6736–6740PubMedCrossRefGoogle Scholar
  51. Steenkamp DJ, Vogt RN (2004) Preparation and utilization of a reagent for the isolation and purification of low-molecular-mass thiols. Anal Biochem 325:21–27PubMedCrossRefGoogle Scholar
  52. Steffek M, Newton GL, Av-Gay Y, Fahey RC (2003) Characterization of Mycobacterium tuberculosis mycothiol S-conjugate amidase. Biochemistry 42:12067–12076PubMedCrossRefGoogle Scholar
  53. Udwary DW, Zeigler L, Asolkar RN, Singan V, Lapidus A, Fenical W, Jensen PR, Moore BS (2007) Genome sequencing reveals complex secondary metabolome in the marine actinomycete Salinispora tropica. Proc Natl Acad Sci USA 104:10376–10381PubMedCrossRefGoogle Scholar
  54. Unson MD, Newton GL, Davis C, Fahey RC (1998) An immunoassay for the detection and quantitative determination of mycothiol. J Immunol Methods 214:29–39PubMedCrossRefGoogle Scholar
  55. Vetting MW, Yu M, Rendle PM, Blanchard JS (2006) The substrate-induced conformational change of Mycobacterium tuberculosis mycothiol synthase. J Biol Chem 281:2795–2802PubMedCrossRefGoogle Scholar
  56. Walsh C (2003) Antibiotics: actions, origins, resistance. ASM Press, Washington, DCGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Gerald L. Newton
    • 1
  • Paul R. Jensen
    • 2
  • John B. MacMillan
    • 3
  • William Fenical
    • 2
  • Robert C. Fahey
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of CaliforniaSan Diego, La JollaUSA
  2. 2.Center for Marine Biotechnology and Biomedicine, Scripps Institution of OceanographyUniversity of CaliforniaSan Diego, La JollaUSA
  3. 3.Department of BiochemistryUT Southwestern Medical CenterDallasUSA

Personalised recommendations