Skip to main content
SpringerLink
Log in

Identification and quantification of mycothiol in Actinobacteria by a novel enzymatic method

  • Methods and Protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Mycothiol (MSH) was reported to be the dominant low molecular weight thiol in members of the Actinobacteria. In this study, a simple, fast, and sensitive method for qualitative and quantitative determination of MSH molecules was developed based on maleylpyruvate isomerase (MPI) from Corynebacterium glutamicum. The principle of this method is that the activity of MPI from C. glutamicum was dependent on MSH molecules. It was found that this MPI activity displayed a linear response (R 2 = 0.9928) at MSH amounts ranging from 0.12 to 3.98 pmol in the defined assay system. This observation was applied to calculate the MSH levels, and the newly developed method was compared with thiol-specific fluorescent-labeling high-performance liquid chromatography method. Forty-eight genera of Actinobacteria were screened for MSH and 43 genera were reported for MSH occurrence, and the MSH levels in Actinobacteria were determined to be 0.01 to 9.69 μmol/g of residual dry cell weight.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Buchmeier NA, Newton GL, Koledin T, Fahey RC (2003) Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. Mol Microbiol 47:1723–1732

    Article  CAS  Google Scholar 

  • den Hengst CD, Buttner MJ (2008) Redox control in actinobacteria. Biochim Biophys Acta 1780:1201–1216

    Google Scholar 

  • Fahey RC, Newton GL (1987) Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography. Methods Enzymol 143:85–96

    Article  CAS  Google Scholar 

  • Feng J, Che Y, Milse J, Yin YJ, Liu L, Ruckert C, Shen XH, Qi SW, Kalinowski J, Liu SJ (2006) The gene ncgl2918 encodes a novel maleylpyruvate isomerase that needs mycothiol as cofactor and links mycothiol biosynthesis and gentisate assimilation in Corynebacterium glutamicum. J Biol Chem 281:10778–10785

    Article  CAS  Google Scholar 

  • Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, Dusch N, Eggeling L, Eikmanns BJ, Gaigalat L, Goesmann A, Hartmann M, Huthmacher K, Kramer R, Linke B, McHardy AC, Meyer F, Mockel B, Pfefferle W, Puhler A, Rey DA, Ruckert C, Rupp O, Sahm H, Wendisch VF, Wiegrabe I, Tauch A (2003) The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins. J Biotechnol 104:5–25

    Article  CAS  Google Scholar 

  • Lack L (1959) The enzymic oxidation of gentisic acid. Biochim Biophys Acta 34:117–123

    Article  CAS  Google Scholar 

  • Larkin MJ, Kulakov LA, Allen CC (2005) Biodegradation and Rhodococcus—masters of catabolic versatility. Curr Opin Biotechnol 16:282–290

    Article  CAS  Google Scholar 

  • Meister A, Anderson ME (1983) Glutathione. Ann Rev Biochem 52:711–760

    Article  CAS  Google Scholar 

  • Newton GL, Fahey RC (2002) Mycothiol biochemistry. Arch Microbiol 178:388–394

    Article  CAS  Google Scholar 

  • Newton GL, Fahey RC, Cohen G, Aharonowitz Y (1993) Low-molecular-weight thiols in Streptomycetes and their potential role as antioxidants. J Bacteriol 175:2734–2742

    CAS  Google Scholar 

  • 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 230:821–825

    Article  CAS  Google Scholar 

  • Newton GL, Arnold K, Price MS, Sherrill C, Delcardayre SB, Aharonowitz Y, Cohen G, Davies J, Fahey RC, Davis C (1996) Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J Bacteriol 178:1990–1995

    CAS  Google Scholar 

  • Newton GL, Unson MD, Anderberg SJ, Aguilera JA, Oh NN, delCardayre SB, Av-Gay Y, Fahey RC (1999) Characterization of Mycobacterium smegmatis mutants defective in 1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside and mycothiol biosynthesis. Biochem Biophys Res Commun 255:239–244

    Article  CAS  Google Scholar 

  • Newton GL, Av-Gay Y, Fahey RC (2000) A novel mycothiol-dependent detoxification pathway in mycobacteria involving mycothiol S-conjugate amidase. Biochemistry 39:10739–10746

    Article  CAS  Google Scholar 

  • Newton GL, Buchmeier N, Fahey RC (2008) Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria. Microbiol Mol Biol Rev 72:471–494

    Article  CAS  Google Scholar 

  • 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 46:3348–3355

    Article  CAS  Google Scholar 

  • Shen XH, Liu SJ (2005) Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum. Sci China C Life Sci 48:241–249

    CAS  Google Scholar 

  • Shen XH, Jiang CY, Huang Y, Liu ZP, Liu SJ (2005) Functional identification of novel genes involved in the glutathione-independent gentisate pathway in Corynebacterium glutamicum. Appl Environ Microbiol 71:3442–3452

    Article  CAS  Google Scholar 

  • Shrout PE, Fleiss JL (1979) Intraclass correlations: uses in assessing rater reliability. Psychol Bull 86:420–428

    Article  CAS  Google Scholar 

  • Spies HS, Steenkamp DJ (1994) Thiols of intracellular pathogens. Identification of ovothiol A in Leishmania donovani and structural analysis of a novel thiol from Mycobacterium bovis. Eur J Biochem 224:203–213

    Article  CAS  Google Scholar 

  • Tarnowski GS, Barclay RK, Mountain IM, Nakamura M, Satterwhite HG, Solney EM (1965) Determination of acid-soluble thiols and disulfides in transplanted animal tumors. Arch Biochem Biophys 110:210–216

    Article  CAS  Google Scholar 

  • Unson MD, Newton GL, Davis C, Fahey RC (1998) An immunoassay for the detection and quantitative determination of mycothiol. J Immunol Methods 214:29–39

    Article  CAS  Google Scholar 

  • Unson MD, Newton GL, Arnold KF, Davis CE, Fahey RC (1999) Improved methods for immunoassay of mycothiol. J Clin Microbiol 37:2153–2157

    CAS  Google Scholar 

  • Wang R, Yin YJ, Wang F, Li M, Zhang HM, Zhang JP, Liu SJ, Chang WR (2007) Crystal structures and site-directed mutagenesis of a mycothiol-dependent enzyme reveal a novel folding and molecular basis for mycothiol-mediated maleylpyruvate isomerization. J Biol Chem 282:16288–16294

    Article  CAS  Google Scholar 

  • Weber T, Welzel K, Pelzer S, Vente A, Wohlleben W (2003) Exploiting the genetic potential of polyketide producing streptomycetes. J Biotechnol 106:221–232

    Article  CAS  Google Scholar 

  • Zhou NY, Fuenmayor SL, Williams PA (2001) nag genes of Ralstonia (formerly Pseudomonas) sp. strain U2 encoding enzymes for gentisate catabolism. J Bacteriol 183:700–708

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is supported by grants from the National Natural Science Foundation of China (30725001). Technical support from Prof. Y-S. Che’s laboratory at the IMCAS (Beijing) and assistance in MSH purification by Ms. Ying Lao are greatly acknowledged.

Author information

Authors and Affiliations

  1. State Key Laboratory of Microbial Resources, Beijing, 100101, People’s Republic of China

    Ya-Jie Yin, Bao-Jun Wang, Cheng-Ying Jiang, Yuan-Ming Luo & Shuang-Jiang Liu

  2. Environmental Microbiology and Biotechnology Research Center, Beijing, 100101, People’s Republic of China

    Ya-Jie Yin, Bao-Jun Wang, Cheng-Ying Jiang & Shuang-Jiang Liu

  3. Environmental Protection Research Institute of Light Industry, Beijing, 100089, People’s Republic of China

    Jing-Hua Jin

  4. Institute of Microbiology, Chinese Academy of Sciences, Bei-Chen-Xi-Lu No. 1, Chao-Yang District, Beijing, 100101, People’s Republic of China

    Shuang-Jiang Liu

Authors
  1. Ya-Jie Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bao-Jun Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cheng-Ying Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuan-Ming Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jing-Hua Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shuang-Jiang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shuang-Jiang Liu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yin, YJ., Wang, BJ., Jiang, CY. et al. Identification and quantification of mycothiol in Actinobacteria by a novel enzymatic method. Appl Microbiol Biotechnol 88, 1393–1401 (2010). https://doi.org/10.1007/s00253-010-2918-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-2918-3

Keywords

Search

Navigation