Improving a natural enzyme activity through incorporation of unnatural amino acids

The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast

turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the

naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and

to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate

binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with

unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino

acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl

group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product

release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged

4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient

catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to

the difficulty in improving this native activity through screening hundreds of thousands of mutants with

natural amino acids. These results demonstrate that designer amino acids provide easy access to new

and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.