Identification of amino acid residues essential for high aflatoxin B1-8,9-epoxide conjugation activity in alpha class glutathione S-transferases through site-directed mutagenesis.
Mice constitutively express glutathione S-transferase mGSTA3-3 in liver. This isoform possesses uniquely high conjugating activity toward aflatoxin B1-8,9-epoxide (AFBO), thereby protecting mice from aflatoxin B1-induced hepatocarcinogenicity. In contrast, rats constitutively express a closely related GST isoenzyme, rGSTA3-3, with low AFBO activity and, therefore, are sensitive to aflatoxin B1 exposure. Although the two GSTs share 86% sequence identity and have similar catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB), they have an approximately 1000-fold difference in catalytic activity toward AFBO. To identify amino acids that confer high activity toward AFBO, non-conserved rGSTA3-3 residues were replaced with mGSTA3-3 residues in two regions believed to form the substrate binding site. Twenty-one mutant rGSTA3-3 enzymes were generated by site-directed mutagenesis using combinations of nine different residues. Except for the E208D mutant, single mutations of rGSTA3-3 produced enzymes with no detectable AFBO activity. Generally, AFBO conjugation activity increased in additive fashion as mGSTA3-3 residues were introduced into the rGSTA3-3 enzyme with the six site mutant E104I/H108Y/Y111H/L207F/E208D/V217K displaying the highest AFBO activity (40 nmol/mg/min) of all the mutant enzymes. When this mutant enzyme was further modified by three additional substitutions (D103E/I105M/V106I) AFBO conjugation activity decreased 14-fold to 2. 8 nmol/mg/min. Although wild-type mGSTA3-3 AFBO conjugation activity (265 nmol/mg/min) could not be obtained by our rGSTA3-3 mutants, we were able to identify six mGSTA3-3 residues; Ile104, Tyr108, His111, Phe207, Asp208, and Lys217 that, when collectively substituted into rGSTA3-3, substantially increased (>200-fold) glutathione conjugation activity toward AFBO.
Van Ness
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Department of Environmental Health, University of Washington, Seattle, Washington 98195, USA.
McHugh
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Bammler
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Eaton
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D016604:Aflatoxin B1 / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004137:Dinitrochlorobenzene / Q000378:metabolism; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology; D004976:Ethacrynic Acid / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
D004137:Dinitrochlorobenzene; D000939:Epitopes; C006426:aflatoxin B1-2,3-oxide; D016604:Aflatoxin B1; D005982:Glutathione Transferase; D004976:Ethacrynic Acid