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.
Glutathione S-transferase (GST) was purified from Escherichia coli K-12, and its N-terminal sequence was determined to be MKLFYKPGAXSLAS. The gene encoding this sequence was cloned and mapped at 1731-1732 kilobases on the E. coli gene map. It encoded a polypeptide of 201 amino acid residues with a calculated molecular weight of 22,860. The overexpressed product of the gene was confirmed to have GST activity toward 1-chloro-2,4-dinitrobenzene and ethacrynic acid and GSH-dependent peroxidase activity toward cumene hydroperoxide. The relative molecular mass of the gene product was determined to be 40,000 by gel chromatography and 25,000 by SDS-polyacrylamide gel electrophoresis, indicating a homodimeric structure. The deduced amino acid sequence was 54% identical with that of Proteus mirabilis GST. Although the homologies between the GSTs from E. coli and mammals were low, many of the residues assigned to be important for the enzymatic function or structure in mammalian cytosolic GSTs were found to be conserved in E. coli GST. Therefore, E. coli GST is considered to have diverged from the same ancestor with other cytosolic GSTs. A specific tyrosyl residue in the vicinity of the N terminus is conserved in all of the known cytosolic GSTs and has been shown to function as a catalytic residue in alpha, mu, and pi class GSTs from mammals. Although Tyr5 in E. coli GST appeared to be the counterpart of the catalytic residue, its replacement with phenylalanine did not significantly affect the enzymatic activity. Therefore, this apparently conserved tyrosyl residue is not essential for catalytic activity in E. coli GST.
Nishida
M
M
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Kong
K H
KH
Inoue
H
H
Takahashi
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D014443:Tyrosine; D005982:Glutathione Transferase