Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3bromo-2-methylpropanoate and ethyl 3phenylbutyrate.
Directed evolution combined with saturation mutagenesis identified six different point mutations that each moderately increases the enantioselectivity of an esterase from Pseudomonas fluorescens (PFE) towards either of two chiral synthons. Directed evolution identified a Thr230Ile mutation that increased the enantioselectivity from 12 to 19 towards methyl (S)-3-bromo-2-methylpropanoate. Saturation mutagenesis at Thr230 identified another mutant, Thr230Pro, with higher-than-wild-type enantioselectivity (E=17). Previous directed evolution identified mutants Asp158Asn and Leu181Gln that increased the enantioselectivity from 3.5 to 5.8 and 6.6, respectively, towards ethyl (R)-3-phenylbutyrate. In this work, saturation mutagenesis identified other mutations that further increase the enantioselectivity to 12 (Asp158Leu) and 10 (Leu181Ser). A homology model of PFE indicates that all mutations lie outside the active site, 12-14 A from the substrate and suggests how the distant mutations might indirectly change the substrate-binding site. Since proteins contain many more residues far from the active site than close to the active site, random mutagenesis is strongly biased in favor of distant mutations. Directed evolution rarely screens all mutations, so it usually finds the distant mutations because they are more common, but probably not the most effective.
Department of Chemistry, McGill University, 801 Sherbrooke St. W., Montréal, Québec H3A 2K6, Canada.
Geoff P
GP
Horsman
Andrew M F
AM
Liu
Erik
E
Henke
Uwe T
UT
Bornscheuer
Romas J
RJ
Kazlauskas
D001665:Binding Sites / Q000235:genetics; D019020:Directed Molecular Evolution; D004950:Esterases / Q000378:metabolism*; D005456:Fluorescent Dyes; D008958:Models, Molecular; D008968:Molecular Conformation; D016296:Mutagenesis; D009154:Mutation / Q000502:physiology*; D010654:Phenylbutyrates / Q000378:metabolism*; D011422:Propionates / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics*; D013237:Stereoisomerism
D005456:Fluorescent Dyes; D010654:Phenylbutyrates; D011422:Propionates; C477061:ethyl 3-phenylbutyrate; C477060:methyl 3-bromo-2-methylpropanoate; D004950:Esterases