Engineering GST M2-2 for High Activity with Indene 1,2-Oxide and Indication of an H-SiteResidue Sustaining Catalytic Promiscuity
2011 (English)In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 412, no 1, 111-120 p.Article in journal (Refereed) Published
The substrate-binding H-site of human glutathionetransferase (GST) M2-2 was subjected to iterative saturation mutagenesis in order to obtain an efficient enzyme with the novel epoxide substrate indene 1,2-oxide. Residues 10, 116, and 210 were targeted, and the activities with the alternative substrates, benzyl isothiocyanate and the prodrug azathioprine, undergoing divergent chemical reactions were monitored for comparison. In general, increased activities were found when the smaller residues Gly, Ser, and Ala replaced the original Thr210. The most active mutant T210G was further mutated at position 116, but no mutant showed enhanced catalytic activity. However, saturation mutagenesis of position 10 identified one double mutant T210G/I10C with 100-fold higher specific activity with indene 1,2-oxide than wild-type GST M2-2. This enhanced epoxide activity of 50 mu mol min(-1) mg(-1) resulted primarily from an increased k(cat) value (70 s(-1)). The specific activity is 24-fold higher than that of wild-type GST M1-1, which is otherwise the most proficient GST enzyme with epoxide substrates. A second double mutant T210G/I10W displayed 30-fold increased activity with azathioprine, 0.56 mu mol min(-1) mg(-1). In both double mutants, the replacement of Ile10 led to narrowed acceptance of alternative substrates. Ile10 is evolutionarily conserved in related class Mu GSTs. Conservation usually indicates preservation of a particular function, and in the Mu class, it would appear that the conservedIle10 is not necessary to maintain catalytic functions but to prevent loss of broad substrate acceptance. In summary, our data underscore the facile transition between alternative substrateselectivity profiles in GSTs by a few mutations.
Place, publisher, year, edition, pages
2011. Vol. 412, no 1, 111-120 p.
protein redesign, active site, conserved residue, saturation mutagenesis, iterative saturation mutagenesis, glutathione transferase, GST M2-2, substrate selectivity, promiscuous
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:uu:diva-149325DOI: 10.1016/j.jmb.2011.07.039ISI: 000294523300011OAI: oai:DiVA.org:uu-149325DiVA: diva2:404546