Improving a natural enzyme activity through incorporation of unnatural amino acids
The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fastturnover rates and are thought to be near to their evolutionary limit for this activity. To test whether thenaturally evolved turnover rate could be improved through the incorporation of unnatural amino acids andto probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substratebinding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 withunnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine aminoacids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxylgroup of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting productrelease step of substrate turnover as a result of its electrostatic repulsion of the negatively charged4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficientcatalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast tothe difficulty in improving this native activity through screening hundreds of thousands of mutants withnatural amino acids. These results demonstrate that designer amino acids provide easy access to newand valuable sequence and functional space for the engineering and evolution of existing enzyme functions.
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