PMID:9576913
| Citation |
Yano, T, Oue, S and Kagamiyama, H (1998) Directed evolution of an aspartate aminotransferase with new substrate specificities. Proc. Natl. Acad. Sci. U.S.A. 95:5511-5 |
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| Abstract |
The substrate specificity of aspartate aminotransferase was successfully modified by directed molecular evolution using a combination of DNA shuffling and selection in an auxotrophic Escherichia coli strain. After five rounds of selection, one of the evolved mutants showed a 10(5)-fold increase in the catalytic efficiency (kcat/Km) for beta-branched amino and 2-oxo acids and a 30-fold decrease in that for the native substrates compared with the wild-type enzyme. The mutant had 13 amino acid substitutions, 6 of which contributed 80-90% to the total effect. Five of these six substitutions were conserved among the five mutants that showed the highest activity for beta-branched substrates. Interestingly, only one of the six functionally important residues is located within a distance of direct interaction with the substrate, supporting the idea that rational design of the substrate specificity of an enzyme is very difficult. The present results show that directed molecular evolution is a powerful technique for enzyme redesign if an adequate selection system is applied. |
| Links | |
| Keywords |
Amino Acid Sequence; Aspartate Aminotransferases/genetics; Aspartate Aminotransferases/metabolism; Directed Molecular Evolution; Escherichia coli/enzymology; Kinetics; Models, Molecular; Molecular Sequence Data; Substrate Specificity |
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