Molecular cloning, expression and characterization of a novel class glutathione S-transferase from the fungus Cunninghamella elegans.
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.
Cha
Chang-Jun
CJ
Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, U.S.A.
Kim
Seong-Jae
SJ
Kim
Yong-Hak
YH
Stingley
Robin
R
Cerniglia
Carl E
CE
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics; D003001:Cloning, Molecular; D020093:Cunninghamella / Q000201:enzymology / Q000235:genetics*; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004137:Dinitrochlorobenzene; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005982:Glutathione Transferase