Identification of Residues in Glutathione Transferase Capable of Driving Functional Diversification in Evolution

Abstract

Evolution of protein function can be driven by positive selection of advantageous nonsynonymous codon mutations that arise following gene duplication. By observing the presence and degree of site-specific positive selection for change between divergent paralogs, residue positions responsible for functional changes can be identified. We applied this analysis to genes encoding Mu class glutathione transferases, which differ widely in substrate specificities. Approximately 3% of the amino acid residue positions, both near to and distant from the active site, are under statistically significant positive selection for change. Relevant human glutathione transferase (GST) M1-1 and GST M2-2 codons were mutated. A chemically conservative threonine to serine mutation in GST M2-2 elicited a 1,000-fold increase in specific activity with the GST M1-1-specific substrate trans-stilbene oxide and a 30-fold increase with the alternative epoxide substrates styrene oxide and nitrophenyl glycidol. The reverse mutation in GST M1-1 resulted in reciprocal decreases in activity. Thus, identification of hypervariable codon positions can be a powerful aid in the redesign of protein function, lessening the requirement for extensive mutagenesis or structural knowledge and sometimes suggesting mutations that would otherwise be considered functionally conservative.

Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution

“…Specific activities with racemic tSBO and SO were determined as described in ref. 5, with the exception that the assay buffer used with tSBO was 100 mM Tris⅐HCl, pH 7.2. Specific activities with CDNB (23), cyanoDMNG (23), NPG (5), and aminochrome (24) were determined by spectrophotometric assays at 30°C under standard conditions.…”

“…The PCR products were purified, ligated, and transformed as described in ref. 5. Expression and purification of GST M2-2 variants was carried out as described in refs.…”

“…GST M2-2 mutants were constructed by PCRs as described in ref. 5. All primers were 24 nucleotides in length and phosphorylated at the 5Ј end.…”

“…In a previous study, we used evolutionary rate analysis to identify a number of hypervariable positions in the Mu-class sequences (5); such positions are under positive selection for change and possibly contribute to functional variations among the divergent paralogues. The hypervariable residue 210 differs between human GST M1-1 and GST M2-2, two enzymes that display distinct differences in substrate-activity profiles.…”

Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum

“…GSTs function primarily as detoxification enzymes, generally rendering the resultant products more water soluble (nonreactive conjugate), thereby facilitating excretion. The catalytic versatility and diversity of GSTs can be attributed to the nonspecific nature of the hydrophobic substrate binding site (H-site), and the extensive gene duplication and divergence that has occurred in this superfamily [2,3]. The soluble GSTs in metazoans are divided into eight classes (alpha, kappa, mu, pi, sigma, theta, omega and zeta) based on sequence identity, immunological and kinetic properties [1,4,5].…”