Molecular basis for the high enantioselectivity of the Ser47Leu variant of Candida antarctica lipase B toward (R)-But-3-yn-2-ol

Abstract

It is known that Candida antarctica lipase B (CAL-B) exhibits low enantioselectivity towards sec-alcohols bearing small substituents such as (±)-but-3-yn-2-ol (E = 5.4) or (±)-butan-2-ol (E = 7), although it possesses high enantioselectivity towards various chiral sec-alcohols. In a previous study, we reported that the Ser47Asn variant, prepared based on the sequence of a homologous enzyme (lipase from Pseudozyma brasiliensis GHG001, PBL), exhibits high enantioselectivity towards (R)-but-3-yn-2-ol (E > 200). However, the molecular basis of high enantioselectivity was unclear because the residue is quite distant from the substituents, making it challenging to establish any interaction. In this study, we prepared a series of variants at the Ser47 residue to understand the molecular basis. We introduced Asp, Glu, Gln, and Leu at the position 47 and found that the Ser47Leu variant also exhibits high enantioselectivity (E > 200). We conducted a molecular dynamics study to elucidate its high enantioselectivity. The introduced leucine shifted the side chain of Trp104, which is a component for constructing the binding pocket for medium substituents, thereby interfering with the binding of the larger substituent of the slow enantiomer.