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. 2002 Dec 1;368(Pt 2):589–595. doi: 10.1042/BJ20020400

Molecular cloning, expression and characterization of a novel class glutathione S-transferase from the fungus Cunninghamella elegans.

Chang-Jun Cha 1, Seong-Jae Kim 1, Yong-Hak Kim 1, Robin Stingley 1, Carl E Cerniglia 1
PMCID: PMC1223007  PMID: 12196209

Abstract

The structural gene for glutathione S-transferase (CeGST1-1) in the fungus Cunninghamella elegans was cloned by screening a cDNA library using a degenerate oligonucleotide probe based on the N-terminal sequence of the purified protein. Open reading frame analysis indicated that the cegst1 gene encodes a protein of 210 amino acid residues. The deduced amino acid sequence showed 25% sequence identity with the sequence of the Pi-class GST from Danio rerio (zebrafish). Similarity was also shown with the Alpha-class GST from Fasciola hepatica (liver fluke; 23% identity), the Mu class from Mus musculus (22%) and the Sigma class from Ommastrephes sloani (squid; 21%). Further screening of a cDNA library with the cegst1 gene probe revealed the presence of another GST isoenzyme (CeGST2-2) in this fungus, which shows 84% sequence identity with CeGST1-1 at the amino acid level. Reverse transcription PCR revealed that cegst2 was also expressed at the mRNA level in the fungus C. elegans. Both cegst genes were overexpressed in Escherichia coli using the expression vector pQE51, displaying specific activities with 1-chloro-2,4-dinitrobenzene of 2.04 and 0.75 micromol/min per mg of protein respectively. Both enzymes exhibited a similar substrate specificity and inhibition profile, indicating that CeGST1-1 and CeGST2-2 belong to the same GST class. Mutagenesis analysis revealed that Tyr(10) in the N-terminal region is essential for catalysis of CeGST1-1. We propose from these results that the CeGSTs are novel Gamma-class GSTs and designated as GSTG1-1 and GSTG2-2 respectively.

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Selected References

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  1. Abourashed E. A., Clark A. M., Hufford C. D. Microbial models of mammalian metabolism of xenobiotics: An updated review. Curr Med Chem. 1999 May;6(5):359–374. [PubMed] [Google Scholar]
  2. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arca P., Rico M., Braña A. F., Villar C. J., Hardisson C., Suárez J. E. Formation of an adduct between fosfomycin and glutathione: a new mechanism of antibiotic resistance in bacteria. Antimicrob Agents Chemother. 1988 Oct;32(10):1552–1556. doi: 10.1128/aac.32.10.1552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Armstrong R. N. Glutathione S-transferases: structure and mechanism of an archetypical detoxication enzyme. Adv Enzymol Relat Areas Mol Biol. 1994;69:1–44. doi: 10.1002/9780470123157.ch1. [DOI] [PubMed] [Google Scholar]
  5. Board P. G., Baker R. T., Chelvanayagam G., Jermiin L. S. Zeta, a novel class of glutathione transferases in a range of species from plants to humans. Biochem J. 1997 Dec 15;328(Pt 3):929–935. doi: 10.1042/bj3280929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Board P. G., Coggan M., Chelvanayagam G., Easteal S., Jermiin L. S., Schulte G. K., Danley D. E., Hoth L. R., Griffor M. C., Kamath A. V. Identification, characterization, and crystal structure of the Omega class glutathione transferases. J Biol Chem. 2000 Aug 11;275(32):24798–24806. doi: 10.1074/jbc.M001706200. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Casalone E., Di Ilio C., Federici G., Polsinelli M. Glutathione and glutathione metabolizing enzymes in yeasts. Antonie Van Leeuwenhoek. 1988;54(4):367–375. doi: 10.1007/BF00393527. [DOI] [PubMed] [Google Scholar]
  9. Cha C. J., Coles B. F., Cerniglia C. E. Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans. FEMS Microbiol Lett. 2001 Sep 25;203(2):257–261. doi: 10.1111/j.1574-6968.2001.tb10850.x. [DOI] [PubMed] [Google Scholar]
  10. Choi J. H., Lou W., Vancura A. A novel membrane-bound glutathione S-transferase functions in the stationary phase of the yeast Saccharomyces cerevisiae. J Biol Chem. 1998 Nov 6;273(45):29915–29922. doi: 10.1074/jbc.273.45.29915. [DOI] [PubMed] [Google Scholar]
  11. Dirr H., Reinemer P., Huber R. X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function. Eur J Biochem. 1994 Mar 15;220(3):645–661. doi: 10.1111/j.1432-1033.1994.tb18666.x. [DOI] [PubMed] [Google Scholar]
  12. Dowd C. A., Buckley C. M., Sheehan D. Glutathione S-transferases from the white-rot fungus, Phanerochaete chrysosporium. Biochem J. 1997 May 15;324(Pt 1):243–248. doi: 10.1042/bj3240243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dowd C. A., Sheehan D. Variable expression of glutathione S-transferase isoenzymes in the fungus, Mucor circinelloides. FEMS Microbiol Lett. 1999 Jan 1;170(1):13–17. doi: 10.1111/j.1574-6968.1999.tb13349.x. [DOI] [PubMed] [Google Scholar]
  14. Eaton D. L., Bammler T. K. Concise review of the glutathione S-transferases and their significance to toxicology. Toxicol Sci. 1999 Jun;49(2):156–164. doi: 10.1093/toxsci/49.2.156. [DOI] [PubMed] [Google Scholar]
  15. Habig W. H., Jakoby W. B. Assays for differentiation of glutathione S-transferases. Methods Enzymol. 1981;77:398–405. doi: 10.1016/s0076-6879(81)77053-8. [DOI] [PubMed] [Google Scholar]
  16. Hayes J. D., Pulford D. J. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol. 1995;30(6):445–600. doi: 10.3109/10409239509083491. [DOI] [PubMed] [Google Scholar]
  17. Iizuka M., Inoue Y., Murata K., Kimura A. Purification and some properties of glutathione S-transferase from Escherichia coli B. J Bacteriol. 1989 Nov;171(11):6039–6042. doi: 10.1128/jb.171.11.6039-6042.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ji X., von Rosenvinge E. C., Johnson W. W., Tomarev S. I., Piatigorsky J., Armstrong R. N., Gilliland G. L. Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods. Biochemistry. 1995 Apr 25;34(16):5317–5328. doi: 10.1021/bi00016a003. [DOI] [PubMed] [Google Scholar]
  19. Ketterer B. A bird's eye view of the glutathione transferase field. Chem Biol Interact. 2001 Oct 25;138(1):27–42. doi: 10.1016/s0009-2797(01)00277-0. [DOI] [PubMed] [Google Scholar]
  20. Kim H. G., Park K. N., Cho Y. W., Park E. H., Fuchs J. A., Lim C. J. Characterization and regulation of glutathione S-transferase gene from Schizosaccharomyces pombe. Biochim Biophys Acta. 2001 Aug 30;1520(2):179–185. doi: 10.1016/s0167-4781(01)00265-2. [DOI] [PubMed] [Google Scholar]
  21. Kohler-Staub D., Leisinger T. Dichloromethane dehalogenase of Hyphomicrobium sp. strain DM2. J Bacteriol. 1985 May;162(2):676–681. doi: 10.1128/jb.162.2.676-681.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kong K. H., Takasu K., Inoue H., Takahashi K. Tyrosine-7 in human class Pi glutathione S-transferase is important for lowering the pKa of the thiol group of glutathione in the enzyme-glutathione complex. Biochem Biophys Res Commun. 1992 Apr 15;184(1):194–197. doi: 10.1016/0006-291x(92)91177-r. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Liu S., Zhang P., Ji X., Johnson W. W., Gilliland G. L., Armstrong R. N. Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3-3 of glutathione S-transferase. J Biol Chem. 1992 Mar 5;267(7):4296–4299. [PubMed] [Google Scholar]
  25. Lloyd-Jones G., Lau P. C. Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons. Appl Environ Microbiol. 1997 Aug;63(8):3286–3290. doi: 10.1128/aem.63.8.3286-3290.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mannervik B., Alin P., Guthenberg C., Jensson H., Tahir M. K., Warholm M., Jörnvall H. Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7202–7206. doi: 10.1073/pnas.82.21.7202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nishida M., Kong K. H., Inoue H., Takahashi K. Molecular cloning and site-directed mutagenesis of glutathione S-transferase from Escherichia coli. The conserved tyrosyl residue near the N terminus is not essential for catalysis. J Biol Chem. 1994 Dec 23;269(51):32536–32541. [PubMed] [Google Scholar]
  28. Pemble S. E., Taylor J. B. An evolutionary perspective on glutathione transferases inferred from class-theta glutathione transferase cDNA sequences. Biochem J. 1992 Nov 1;287(Pt 3):957–963. doi: 10.1042/bj2870957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pemble S. E., Wardle A. F., Taylor J. B. Glutathione S-transferase class Kappa: characterization by the cloning of rat mitochondrial GST and identification of a human homologue. Biochem J. 1996 Nov 1;319(Pt 3):749–754. doi: 10.1042/bj3190749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ranson H., Cornel A. J., Fournier D., Vaughan A., Collins F. H., Hemingway J. Cloning and localization of a glutathione S-transferase class I gene from Anopheles gambiae. J Biol Chem. 1997 Feb 28;272(9):5464–5468. doi: 10.1074/jbc.272.9.5464. [DOI] [PubMed] [Google Scholar]
  31. Ranson H., Prapanthadara L. a., Hemingway J. Cloning and characterization of two glutathione S-transferases from a DDT-resistant strain of Anopheles gambiae. Biochem J. 1997 May 15;324(Pt 1):97–102. doi: 10.1042/bj3240097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ranson H., Rossiter L., Ortelli F., Jensen B., Wang X., Roth C. W., Collins F. H., Hemingway J. Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae. Biochem J. 2001 Oct 15;359(Pt 2):295–304. doi: 10.1042/0264-6021:3590295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sheehan D., Casey J. P. Evidence for alpha and Mu class glutathione S-transferases in a number of fungal species. Comp Biochem Physiol B. 1993 Jan;104(1):7–13. doi: 10.1016/0305-0491(93)90331-x. [DOI] [PubMed] [Google Scholar]
  34. Sheehan D., Casey J. P. Microbial glutathione S-transferases. Comp Biochem Physiol B. 1993 Jan;104(1):1–6. doi: 10.1016/0305-0491(93)90330-8. [DOI] [PubMed] [Google Scholar]
  35. Sheehan D., Meade G., Foley V. M., Dowd C. A. Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem J. 2001 Nov 15;360(Pt 1):1–16. doi: 10.1042/0264-6021:3600001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stenberg G., Board P. G., Mannervik B. Mutation of an evolutionarily conserved tyrosine residue in the active site of a human class Alpha glutathione transferase. FEBS Lett. 1991 Nov 18;293(1-2):153–155. doi: 10.1016/0014-5793(91)81174-7. [DOI] [PubMed] [Google Scholar]
  37. Tamaki H., Kumagai H., Tochikura T. Nucleotide sequence of the yeast glutathione S-transferase cDNA. Biochim Biophys Acta. 1991 Jun 13;1089(2):276–279. doi: 10.1016/0167-4781(91)90025-h. [DOI] [PubMed] [Google Scholar]
  38. Tamaki H., Kumagai H., Tochikura T. Purification and properties of glutathione transferase from Issatchenkia orientalis. J Bacteriol. 1989 Feb;171(2):1173–1177. doi: 10.1128/jb.171.2.1173-1177.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vuilleumier S. Bacterial glutathione S-transferases: what are they good for? J Bacteriol. 1997 Mar;179(5):1431–1441. doi: 10.1128/jb.179.5.1431-1441.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wackett L. P., Gibson D. T. Metabolism of xenobiotic compounds by enzymes in cell extracts of the fungus Cunninghamella elegans. Biochem J. 1982 Jul 1;205(1):117–122. doi: 10.1042/bj2050117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wang R. F., Khan A. A., Cao W. W., Cerniglia C. E. Identification and sequencing of a cDNA encoding 6-phosphogluconate dehydrogenase from a fungus, Cunninghamella elegans and expression of the gene in Escherichia coli. FEMS Microbiol Lett. 1998 Dec 15;169(2):397–402. doi: 10.1111/j.1574-6968.1998.tb13346.x. [DOI] [PubMed] [Google Scholar]
  42. Wilce M. C., Board P. G., Feil S. C., Parker M. W. Crystal structure of a theta-class glutathione transferase. EMBO J. 1995 May 15;14(10):2133–2143. doi: 10.1002/j.1460-2075.1995.tb07207.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhang D., Yang Y., Leakey J. E., Cerniglia C. E. Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics. FEMS Microbiol Lett. 1996 May 1;138(2-3):221–226. doi: 10.1111/j.1574-6968.1996.tb08161.x. [DOI] [PubMed] [Google Scholar]

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