A novel and robust 3-quinuclidinone reductase from Kaistia algarum for efficient synthesis of (R)-3-quinuclidinol without external cofactor

Abstract

A novel ketone reductase KaKR was identified from Kaistia algarum through genome mining with high activity and enantioselectivity toward 3-quinuclidione. Sequence alignment and conserved motifs analysis revealed that KaKR was classified into classical short-chain dehydrogenase/reductase family. KaKR displayed the highest activity at pH 7.0 and 50°C. Substrate spectrum analysis revealed that KaKR exhibited high substrate specificity toward N-heterocyclic ketones. The kcat/KM toward 3-quinuclidione and NADH were 82.8 and 1697 s–1⋅mM–1. KaKR is a robust biocatalyst and could tolerate high concentrations of 3-quinuclidione. Without assistance of external cofactor, as high as 5.0 M 3-quinuclidione could be completely and enantioselectivity reduced into (R)-quinuclidinol with ee of >99.9%, S/C of 42.6 g⋅g–1 and space-time yield of 1027 g⋅L–1⋅d–1. Alanine scanning was performed to elucidate the molecular mechanism of the high substrate specificity and enantioselectivity. Two critical residues, V94 and E198, were identified with important roles in determining the catalytic efficiency and enantioselectivity. This study provides a novel and robust ketone reductase, and establishes an efficient and economic bioprocess for the synthesis of (R)-3-quinuclidinol without addition of external cofactor.