Inverting the Enantiopreference of Nitrilase‐Catalyzed Desymmetric Hydrolysis of Prochiral Dinitriles by Reshaping the Binding Pocket with a Mirror‐Image Strategy

Abstract

A mirror‐image strategy, that is, symmetry analysis of the substrate‐binding pocket, was applied to identify two key amino acid residues W170 and V198 that possibly modulate the enantiopreference of a nitrilase from Synechocystis sp. PCC6803 towards 3‐isobutyl glutaronitrile (1 a). Exchange of these two residues resulted in the enantiopreference inversion (S, 90 % ee to R, 47 % ee). By further reshaping the substrate‐binding pocket via routine site‐saturation and combinatorial mutagenesis, variant E8 with higher activity and stereoselectivity (99 % ee, R) was obtained. The mutant enzyme was applied in the preparation of optically pure (R)‐3‐isobutyl‐4‐cyanobutanoic acid ((R)‐2 a) and showed similar stereopreference inversion towards a series of 3‐substituted glutaronitriles. This study may offer a general strategy to switch the stereopreference of other nitrilases and other enzymes toward the desymmetric reactions of prochiral substrates with two identical reactive functional groups.

Engineering of reaction specificity, enantioselectivity, and catalytic activity of nitrilase for highly efficient synthesis of pregabalin precursor

“…In contrast, pathway B was the main pathway. In general, larger difference between Dso and Dno (Dso-Dno) would lead to the reduction of amide formation (Jiang et al, 2017;Yu et al, 2020). Moreover, steric hindrance, stereochemical and electronic properties of the R group in the substrate, and temperature, pH of the reaction environment would affect the reaction specificity of nitrilases (Dai et al, 2022;Fernandes et al, 2006).…”

“…Nitrilases (EC 3.5.5.1) catalyze the hydrolysis of nitriles to the corresponding carboxylic acid and ammonia (Shen et al, 2020). Over the past decades, nitrilase-mediated bioprocess has attracted much attention for manufacturing value-added carboxylic acids (Desantis et al, 2003;Vergne-Vaxelaire et al, 2013;Xue et al, 2015), including the building blocks of pharmaceuticals (Yu et al, 2020), pesticides (Dai et al, 2022), feed additives (Gong et al, 2018;Teepakorn et al, 2021) and polymers (Ben-Bassat et al, 2008). The nitrilasecatalyzed chemoenzymatic route for pregabalin began with the condensation of isovaleraldehyde and cyanoacetate, followed by cyanation to give isobutylsuccinonitrile (IBSN).…”

Stereodivergent Synthesis of Epoxides and Oxazolidinones via the Halohydrin Dehalogenase-Catalyzed Desymmetrization Strategy

“…Catalytic stereoselective desymmetrization that can generate chiral compounds by eliminating one or more elements of symmetry of the substrates has been widely recognized as one of the most attractive and important strategies in asymmetric synthesis. It is therefore no surprise that many chemical desymmetrization − and biocatalytic desymmetrization − methods have been established to date.…”

Going Beyond the Local Catalytic Activity Space of Chitinase Using a Simulation-Based Iterative Saturation Mutagenesis Strategy

“…Enzyme engineering is a powerful tool for the design of proteins or enzymes with higher activity or selectivity by changing the sequence of amino acids through mutations introduced via recombinant DNA technology. During the past decade, enzyme engineering has demonstrated its usefulness and applicability in altering and improving substrate specificity, , catalytic activity, , enantioselectivity, , stability, , and de novo enzyme design. , Directed evolution and rational design are widely used approaches for enzyme engineering. Directed laboratory evolution is the process of generating mutant libraries via the natural evolution process involving iterative rounds of gene mutation and identifying efficient mutants with better properties.…”