Novel Alcohol Dehydrogenase CgADH from Candida glabrata for Stereocomplementary Reduction of Bulky–Bulky Ketones Featuring Self-Sufficient NADPH Regeneration

Abstract

Bioreduction of ketones with self-sufficient cofactor regeneration is green and sustainable for the synthesis of chiral secondary alcohols. In this study, a novel alcohol dehydrogenase CgADH was identified from Candida glabrata through genome hunting, exhibiting high oxidation and reduction activities. Conserved motif analysis revealed that CgADH belongs to the extended SDR subfamily. CgADH is NADP(H) dependent and displays the highest activity at pH 5.0 and 65 °C. Substrate spectrum analysis indicated that CgADH exhibited high specificity toward 2-propanol and 2,3butanediol. Rational engineering of CgADH was performed to modulate its stereoselectivity in the asymmetric reduction of bulky−bulky ketones. Two stereocomplementary mutants, C244A and V222G/C244N, were obtained with e.e. values of 99.6% (R) or 94.5% (S) toward (4-chlorophenyl)-pyridin-2-ylmethanone, respectively. Moreover, the catalytic efficiency (k cat /K M ) of C244A and V222G/C244N increased by 70-and 25-fold compared with the wild type (WT), respectively. C244A and V222G/ C244N also displayed significantly enhanced catalytic efficiency and stereoselectivity toward diaryl ketones with different substituents. Using isopropanol both as a co-substrate and as a co-solvent, a concise selfsufficient NADPH regeneration system was developed to demonstrate the advantage of CgADH. This study provides evidence of the application potential of the newly identified CgADH in the preparation of enantiopure diaryl alcohols with low byproducts and E factor.

Microbial alcohol dehydrogenases: recent developments and applications in asymmetric synthesis

“…Substrate profiling of C. glabrata ADH (CgADH), a metal independent, NADPH dependent SDR revealed its highest specificity for isopropanol (IPA) oxidation suggesting its potential as a cofactor regeneration enzyme. 115 Wild type CgADH cat-alysed CPMK (45) reduction produced only 86.8% ee of (R)-46 and 0.22 U mg −1 specific activity. To alter the substrate selectivity, CgADH was engineered by SSM at V222 and C244, which were selected from a docking study.…”

Engineering ketoreductases for the enantioselective synthesis of chiral alcohols

“…alkyl, alkoxy, amine), and electron-withdrawing (e.g. nitro, cyano, halogen, trifluoromethyl, carboxylate ester), 48,94,95 and bulky aryl, 96 not to mention other aromatic ring systems. 97 For example, Lactobacillus composti reductase SDR, heterologously expressed in E. coli, catalysed the enantioselective reduction of acetophenone to R-1phenylethanol in 499% ee and 562.8 g L À1 d À1 STY.…”

Resin-Immobilized Palladium Acetate and Alcohol Dehydrogenase for Chemoenzymatic Enantioselective Synthesis of Chiral Diarylmethanols

“…22,23 Besides, alcohol dehydrogenases (ADHs) and ketoreductases (KREDs) are often used for chiral alcohol synthesis by reduction of prochiral ketones. Recent developments in the molecular biology field allowed the emergence of various valuable mutations of ADHs, leading to efficient biocatalysts that can be applied toward bulky−bulky prochiral substrates, which are hardly accepted by the wild-type enzymes, 24 including ADHs from Kluyveromyces polyspora (KpADHs), 18,25 Thermoanaerobacter brockii (TbSADH), 26 Stenotrophomonas maltophilia (CgADH), 27 and Lactobacillus kefiri (LkADH). 28 For example, Zhou, Ni et al achieved the enantioselective reduction of diaryl ketones using native and engineered KpADHs (Scheme 1, eq 2b).…”

“…Enzymatic transformations have emerged as a competitive alternative in asymmetric synthesis due to their high selectivity, renewability, and mild catalytic conditions. , In particular, the advent of directed evolution and rational design technologies has revolutionized the realm of enzyme catalysis, empowering enzymes to proficiently convert many otherwise recalcitrant substrates. − Indeed, the enzymatic enantioselective synthesis of chiral diarylmethanols has now also been documented. − Candida antarctica lipase B (CALB) mutants have been designed with enhanced enantioselectivity in kinetic resolution of diarylmethanol acetates, enabling the synthesis of phenyl-pyridylmethanols in high enantiomeric excess (ee) values (Scheme , eq 2c). , Besides, alcohol dehydrogenases (ADHs) and ketoreductases (KREDs) are often used for chiral alcohol synthesis by reduction of prochiral ketones. Recent developments in the molecular biology field allowed the emergence of various valuable mutations of ADHs, leading to efficient biocatalysts that can be applied toward bulky–bulky prochiral substrates, which are hardly accepted by the wild-type enzymes, including ADHs from Kluyveromyces polyspora ( Kp ADHs), , Thermoanaerobacter brockii ( TbS ADH), Stenotrophomonas maltophilia ( Cg ADH), and Lactobacillus kefiri ( Lk ADH) . For example, Zhou, Ni et al achieved the enantioselective reduction of diaryl ketones using native and engineered Kp ADHs (Scheme , eq 2b) .…”