Sigma-class glutathione transferase from Xenopus laevis: molecular cloning, expression, and site-directed mutagenesis.
The structural gene for glutathione transferase (XlGSTS1-1) in the amphibia Xenopus laevis has been cloned from an embryo library and its nucleotide sequence has been determined. Open reading frame analysis indicated that xlgsts1 gene encodes the smallest protein of sigma class GST so far identified as being composed of only 194 amino acid residues. The recombinant XlGSTS1-1 shows a narrow range of substrate specificity as well as a significantly lower 1-chloro-2,4-dinitrobenzene conjugation capacity than that of squid sigma class GST. To compare the structural and functional differences between the squid and amphibian enzymes, several site-specific mutations were introduced in XlGSTS1-1, i.e., Ser100Asn, Phe102Tyr, Trp143Leu, Phe146Leu, and Trp148Cys. The results obtained indicate that Trp143 and Trp148 are more important determinants for the structural stability of XlGSTS1-1 rather than for its substrate specificity.
Dipartimento di Scienze Biomediche, Università G. D'Annunzio, Chieti, Italy.
Erminia
E
Carletti
Antonella
A
De Luca
Andrea
A
Urbani
Paolo
P
Sacchetta
Carmine
C
Di Ilio
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D049832:Decapodiformes / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment / Q000379:methods; D020539:Sequence Analysis, Protein; D017385:Sequence Homology; D013045:Species Specificity; D013379:Substrate Specificity; D014982:Xenopus laevis / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D005982:Glutathione Transferase