Skip Navigation
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.


The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Your Environment. Your Health.

Publication Detail

Title: 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.

Authors: Van Ness, K P; McHugh, T E; Bammler, T K; Eaton, D L

Published In Toxicol Appl Pharmacol, (1998 Sep)

Abstract: 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.

PubMed ID: 9772212 Exiting the NIEHS site

MeSH Terms: Aflatoxin B1/analogs & derivatives*; Aflatoxin B1/genetics; Aflatoxin B1/metabolism; Amino Acid Sequence; Animals; Binding Sites; Dinitrochlorobenzene/metabolism; Epitopes; Escherichia coli/enzymology; Ethacrynic Acid/metabolism; Glutathione Transferase/genetics; Glutathione Transferase/metabolism*; Mice; Microsomes, Liver/enzymology; Models, Molecular; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed*; Research Support, U.S. Gov't, P.H.S.; Sequence Homology, Amino Acid

to Top