title: Improving a natural enzyme activity through incorporation of unnatural amino acids.
abstract: 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.
authors.affiliation: Commonwealth Scientific and Industrial Research Organization, Black Mountain, Canberra, Australia.
authors.forename: Isaac N
authors.initials: IN
authors.lastname: Ugwumba
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ozawa
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Xu
authors.forename: Fernanda
authors.initials: F
authors.lastname: Ely
authors.forename: Jee-Loon
authors.initials: JL
authors.lastname: Foo
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Herlt
authors.forename: Chris
authors.initials: C
authors.lastname: Coppin
authors.forename: Sue
authors.initials: S
authors.lastname: Brown
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Taylor
authors.forename: David L
authors.initials: DL
authors.lastname: Ollis
authors.forename: Lewis N
authors.initials: LN
authors.lastname: Mander
authors.forename: Gerhard
authors.initials: G
authors.lastname: Schenk
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Dixon
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
authors.forename: John G
authors.initials: JG
authors.lastname: Oakeshott
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Jackson
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D015394:Molecular Structure; D010261:Paraoxon / Q000737:chemistry / Q000378:metabolism*; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D044345:Phosphoric Triester Hydrolases; D010261:Paraoxon