Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids
Ugwumba, Isaac N. and Ozawa, Kiyoshi and Xu, Zhi-Qiang and Ely, Fernanda and Foo, Jee-Loon and Herlt, Anthony J. and Coppin, Chris and Brown, Sue and Taylor, Matthew C. and Ollis, David L. and Mander, Lewis N. and Schenk, Gerhard and Dixon, Nicholas E. and Otting, Gottfried and Oakeshott, John G. and Jackson, Colin J. (2011) Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids. Journal of the American Chemical Society, 133 (2). pp. 326-333. ISSN 0002-7863
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.
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