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  • Title: Redesign of enzyme for improving catalytic activity and enantioselectivity toward poor substrates: manipulation of the transition state.
    Author: Ema T, Nakano Y, Yoshida D, Kamata S, Sakai T.
    Journal: Org Biomol Chem; 2012 Aug 21; 10(31):6299-308. PubMed ID: 22710791.
    Abstract:
    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/π interaction partly enhanced the (R)-enantiomer reactivity, the estimated energy of the CH/π 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.
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