Redesign of enzyme for improving catalytic activity and enantioselectivity toward poor substrates: manipulation of the transition stateElectronic supplementary information (ESI) available: Site-directed mutagenesis, synthesis of 1, lipase-catalyzed reactions of 1, and copies of NMR spectra. See DOI: 10.1039/c2ob25614b

Secondary alcohols having bulky substituents on both sides of the hydroxy group are inherently poor substrates for most lipases. In view of this weakness, we redesigned a Burkholderia cepacia lipase to create a variant with improved enzymatic characteristics. The I287F/I290A double mutant showed a high conversion and a high E value (>200) for a poor substrate for which the wild-type enzyme showed a low conversion and a low E value (5). This enhancement of catalytic activity and enantioselectivity of the variant resulted from the cooperative action of two mutations: Phe287 contributed to both enhancement of the ( R )-enantiomer reactivity and suppression of the ( S )-enantiomer reactivity, while Ala290 created a space to facilitate the acylation of the ( R )-enantiomer. The kinetic constants indicated that the mutations effectively altered the transition state. Substrate mapping analysis strongly suggested that the CH/ interaction partly enhanced the ( R )-enantiomer reactivity, the estimated energy of the CH/ interaction being 0.4 kcal mol 1 . The substrate scope of the I287F/I290A double mutant was broad. This biocatalyst was useful for the dynamic kinetic resolution of a variety of bulky secondary alcohols for which the wild-type enzyme shows little or no activity. Catalytic activity and enantioselectivity of lipase toward poor substrates bearing bulky substituents on both sides have been dramatically improved.