Molecular Basis for the High Activity and Enantioselectivity of the Carbonyl Reductase from Sporobolomyces salmonicolor toward α-Haloacetophenones

Abstract

In an effort to develop a practical method for the synthesis of optically pure 2,2,2-trifluoro-1-phenylethanol, we found that the carbonyl reductase (SSCR) from Sporobolomyces salmonicolor showed excellent activity and enantioselectivity toward the halogenated acetophenones. Especially, SSCR exhibited more than 1000 times higher activity toward α,α,α-trifluoroacetophenone than unsubstituted acetophenone, a strikingly different observation from the previously well-studied alcohol dehydrogenase (LBADH) from Lactobacillus brevis. Enzyme−substrate docking and site-directed mutagenesis studies revealed the molecular basis for the high enzyme activity and enantioselectivity of SSCR toward the αhalogenated acetophenones. The hydrogen bond of the Asn207 side chain with the substrate halogen atom and the XH/π interaction of the substrate phenyl group with the side chains of Ser222/ Thr223 resulted in the formation of the highly reactive conformation of α-halogenated acetophenones in the active site of the enzyme. (S)-2,2,2-Trifluoro-1-phenylethanol was prepared in excellent isolated yield and enantiomeric excess from the reduction of α,α,α-trifluoroacetophenone with mutant T209A. These results suggest that tuning the interactions between the halogen atoms/phenyl group of the substrate and the amino acid residues of the enzyme would lead to valuable mutants for the practical synthesis of β-haloalcohols.

Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

“…These methodologies, including partial least-squares regression, random forest, decision trees, support vector machines, K-nearest neighbors, and neural networks, have been discussed in comprehensive reviews 52 – 57 . Overall, the machine-learning based evolution round was one puzzle piece in a comprehensive enzyme optimization campaign: We further improved the enzyme activity and stability by targeting experimentally determined and literature-derived 31 , 33 – 35 , 58 amino acid positions by applying iterative site saturation mutagenesis 47 . Overall, key mutations for the efficient reduction of 1a as elucidated by kinetic studies, and modeling included active site residues T134V, A238K, M242W and Q245S while changes on the enzyme surface and in the substrate access tunnel more likely contribute to stability of the enzyme under process conditions.…”

Engineered ketoreductase-catalyzed stereoselective reduction of ethyl 2′-ketopantothenate and its analogues: chemoenzymatic synthesis of d-pantothenic acid

“…On the other hand, (R)-PaOEt of merely 24% ee was afforded in the KmCR2-catalyzed reduction of K-PaOEt, although 89% conversion was achieved (entry 4, Table 1). To our delight, complete conversion (>99%) was accomplished in the reduction reaction catalyzed by SSCR (entry 5, Table 1), a ketoreductase originating from Sporobolomyces salmonicolor AKU4429, [23][24][25] and the target (R)-PaOEt was generated in perfect optical purity (>99% ee). We determined the specific activity of the purified SSCR with a N-terminal His 6 -tag against K-PaOEt as 109.9 U mg −1 (entry 1, Table 2).…”

Multienzymatic Cascades and Nanomaterial Scaffolding—A Potential Way Forward for the Efficient Biosynthesis of Novel Chemical Products

“…Over the past 15 years or so there has been the development of at least 4 different biocatalytic routes for the green synthesis of β‐halohydrins. [ 84–87 ] However, one of the more significant limitations of these biocatalytic routes is the absolute requirement for prior oxygen‐functionalization at the desired C─H bond site, limiting the available substrate pool to carbonyl or hydroxylated precursors. To address this latter issue, Cui et al.…”

“…Over the past 15 years or so there has been the development of at least 4 different biocatalytic routes for the green synthesis of 𝛽-halohydrins. [84][85][86][87] However, one of the more significant limitations of these biocatalytic routes is the absolute requirement for prior oxygenfunctionalization at the desired C─H bond site, limiting the available substrate pool to carbonyl or hydroxylated precursors. To address this latter issue, Cui et al exploited the enzymatic promiscuity and powerful oxidation reactions of cytochrome P450s (CYP450, EC 1.14.14.86, monomer) to asymmetrically hydroxylate the C─H bond of 𝛽halohydrocarbons to generate the requisite enantioenriched 𝛽-halohydrins (Scheme 4, E1).…”