Identification of Residues in Glutathione Transferase Capable of Driving Functional Diversification in Evolution: A NOVEL APPROACH TO PROTEIN REDESIGN

Ylva Ivarsson,Aaron J Mackey,Maryam Edalat,William R Pearson,Bengt Mannervik

doi:10.1074/jbc.m211776200

Ylva Ivarsson, Aaron J Mackey + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m211776200

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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.

Highlights

Evolution of protein function can be driven by positive selection of advantageous nonsynonymous codon mutations that arise following gene duplication
Under different models of positive selection [18], residues 130, 210, and 214 were consistently identified as likely to be under positive selection, whereas residues 104, 205, and 206 were less strongly indicated
Expression and Purification—Single, double, and triple mutations were made to human GST M2-2 at positions 210, 130, and 104 (T210S, A130E, F104T), sequentially replacing the original GST M2-2 residues with the corresponding human GST M1-1 residues

Summary

A NOVEL APPROACH TO PROTEIN REDESIGN*

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. Whereas the structure of the G-site is well conserved among GSTs, the H-site varies widely in different classes, leading to differences in substrate selectivities. Previous attempts to rationally redesign the substrate selectivities of GSTs have relied upon structural comparisons of homologous proteins, predicting functionally important H-site. We use an evolutionary approach, asking whether positive selection can be observed within Mu class GST genes, by first observing the naturally occurring mutations at positions identified to be under positive selection and directly testing whether these mutations confer altered substrate selectivity

EXPERIMENTAL PROCEDURES

82 Ϯ 7 181 Ϯ 4 547 Ϯ 12

RESULTS

DISCUSSION