Substrate expansion of Geotrichum candidum alcohol dehydrogenase towards diaryl ketones by mutation

Abstract

Chiral diaryl alcohols, such as (4-chlorophenyl)(pyridin-2-yl)methanol, are important intermediates for pharmaceutical synthesis. However, using alcohol dehydrogenases (ADHs) in the asymmetric reduction of diaryl ketones to produce the corresponding alcohols is challenging due to steric hindrance in the substrate binding pockets of the enzymes. In this study, the steric hindrance of the ADH from Geotrichum candidum NBRC 4597 (G. candidum acetophenone reductase, GcAPRD) was eliminated by simultaneous site-directed mutagenesis of Phe56 (in the large pocket) and Trp288 (in the small pocket). As a result, two double mutants, Phe56Ile/Trp288Ala, and Phe56Ala/Trp288Ala, exhibited much higher specific activities towards 2-(4'-chlorobenzoyl)pyridine (4.5 μmol/min/mg and 3.4 μmol/min/mg, respectively) than the wild type (< 0.2 μmol/min/mg). In whole-cell-catalyzed asymmetric reductions of diaryl ketones, Phe56Ile/Trp288Ala significantly increased the isolated yields, which were over 90% for the reactions of most of the tested substrates. Regarding enantioselectivity, Phe56Ile/Trp288Ala and Phe56Ala/Trp288Ala, and Trp288Ala generally exhibited similar selectivity to produce (R)-alcohols with up to 97% ee. KEY POINTS: • Phe56 in Geotrichum reductase (GcAPRD) was mutated to eliminate steric hindrance. • Mutation at Phe56 increased enzymatic activity and expanded substrate specificity. • Phe56Ile/Trp288Ala showed high activity and (R)-selectivity towards diaryl ketones.

Keywords: Alcohol dehydrogenase; Asymmetric reduction; Chiral diaryl alcohol; Diaryl ketone; Enzyme engineering; Site-directed mutagenesis.

Fig. 1

Mutants constructed and substrates/products used in this study. ^aThe absolute configuration (S/R) is determined by the Cahn–Ingold–Prelog priority rule. Therefore, when the phenyl group enters the large pocket, and the methyl or pyridyl group enters the small pocket, (S)-1b-(S)-11b and (R)-12b-(R)-19b are formed, respectively

Fig. 2

The relative activity of (A) wild type^a and (B) Phe56Ile towards 1a-11a. ^aThe preparative-scale reduction of these substrates by the wild type were published previously, but not those for the relative activity

Fig. 3

Analytical-scale reductions of 1a-11a by GcAPRD Phe56Ile: (A) conversion and (B) enantioselectivity. The reaction conditions are described in the “Materials and methods,” section “Analytical-scale asymmetric reduction of 1a-11a by GcAPRD Phe56Ile”

Fig. 4

Reductions of 12a–19a by GcAPRD mutants: (A) yield and (B) ee for the reaction by Trp288Ala, (C) yield and (D) ee for the reaction by Phe56Ile/Trp288Ala, and (E) yield and (F) ee for the reaction by Phe56Ala/Trp288Ala. The reaction conditions are described in the “Materials and methods,” section “Asymmetric reduction of diaryl ketones by GcAPRD Trp288Ala, Phe56Ile/Trp288Ala, and Phe56Ala/Trp288Ala”