title: Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations.
abstract: Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach--random mutagenesis within the substrate binding site--is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.
authors.affiliation: Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montréal, Québec H3A 2K6, Canada.
authors.forename: Seongsoon
authors.initials: S
authors.lastname: Park
authors.forename: Krista L
authors.initials: KL
authors.lastname: Morley
authors.forename: Geoff P
authors.initials: GP
authors.lastname: Horsman
authors.forename: Mats
authors.initials: M
authors.lastname: Holmquist
authors.forename: Karl
authors.initials: K
authors.lastname: Hult
authors.forename: Romas J
authors.initials: RJ
authors.lastname: Kazlauskas
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D016296:Mutagenesis; D009154:Mutation; D011422:Propionates / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013237:Stereoisomerism
chemical_list: D011422:Propionates; C477060:methyl 3-bromo-2-methylpropanoate; D004950:Esterases