Recombinant expression and molecular insights into the catalytic mechanism of an NADPH-dependent conjugated polyketone reductase for the asymmetric synthesis of (R)-pantolactone

Abstract

(R)-pantolactone is a key chiral intermediate for synthesizing calcium (R)-pantothenate. The commercial synthesis of (R)-pantolactone is performed through the resolution of racemic pantolactone using lactonase-catalyzed enantioselective hydrolysis. The process needs highly toxic hydrogen cyanide and a tedious dynamic kinetic resolution. In this study, we investigated an alternative method to prepare (R)-pantolactone through asymmetric reduction of ketopantolactone (KPL). An NADPH-dependent conjugated polyketone reductase gene from Candida dubliniensis CD36 (CduCPR) was functionally overexpressed in Escherichia coli BL21 (DE3). Recombinant CduCPR belonged to the aldo-keto reductase superfamily, and showed high catalytic activity and stereoselectivity using KPL as the substrate. In a continuous feeding reaction, 200 mM ketopantolactone was reduced to (R)-pantolactone with 98% conversion and 99% enantiomeric excess (e.e.) within 2.0 h. The catalytic mechanism was further investigated. Tyr66 functions as a proton donor following hydrogen transfer from NADPH. Thr30 and His128 are critical residues to bind and orient KPL. Therefore, the recombinant CduCPR from C. dubliniensis exhibited potential application in the asymmetric synthesis of (R)-pantolactone.