Origins of stereoselectivity in evolved ketoreductases

Abstract

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo-and NADP + -bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation. directed evolution | crystallographic structures | molecular dynamics | theozyme | enantioselectivity B iocatalysis is a common method of stereoselective ketone reduction (1). This approach often replaces multistep syntheses and uses renewable, biodegradable, and nontoxic reagents and mild conditions (2). Ketoreductases (KREDs), the most commonly used enzymes in industrial pharmaceutical synthesis (3), reduce a wide range of ketones to alcohols with high chemoselectivity and stereoselectivity. These enzymes have been engineered to synthesize alcohols as intermediates for the production of atorvastatin (Lipitor), montelukast (Singulair), and atazanavir (Reyetaz) (4).Small and almost symmetrical ketones, such as prochiral cyclopentanones, are attractive substrates that are difficult to reduce asymmetrically by chemical methods (5, 6). In particular, the enantiopure chiral alcohols derived from 3-oxacyclopentanone (1) and 3-thiacyclopentanone (2) are used in the synthesis of the pharmaceutical agents fosamprenavir and sulopenem, respectively (Fig. 1). Through a directed evolution (DE) program, Codexis, Inc. engineered a KRED obtained from Lactobacillus kefir for the reduction of 3-thiacyclopentanone (2) for the large-scale production of the antibiotic sulopenem. L. kefir KRED (WT) belongs to the short-chain dehydrogenase/reductase (SDR) family (7,8). Via DE, a variant containing 10 mutati...

Stabilization of Multimeric Proteins via Intersubunit Cyclization

“…Due to this potential and the availability of Leifsonia aquatica ATCC 14665, we chose the highly similar protein LnADH from this strain, which differs by three residues from LsADH, as a study model throughout this work (see Materials and Methods). The protein structure of LnADH is not currently available, but there are a few structures of ADHs that have been solved (16)(17)(18). These structures are identical to each other and bear the typical homotetrameric fold of SDRs.…”

Enhancement of asymmetric bioreduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine to corresponding (S)-enantiomer by fusion of carbonyl reductase and glucose dehydrogenase

“…Compared with chemical synthesis, biocatalytic route has become a subject of considerable interest due to its high chemo-, regio-, and enantioselectivities, and mild reaction conditions and environmental benignity (Lalonde 2016;Ni and Xu 2012;Wohlgemuth 2010). In recent years, enzymatic asymmetric reduction of prochiral ketones for preparation of optically active alcohols has gained increasing favor (Nakamura et al 2003;Nealon et al 2015;Noey et al 2015;Sun et al 2016;Wang et al 2011).…”

Computational modelling of enzymes: predicting and understanding the selectivity of an epoxide hydrolase

“…Computational and statistical methods can predict the effects of mutations on enzyme selectivity from sparse sequence-activity data sets. Statistical and molecular modelling methods (see Chapter 5) have generally been used to model enantioselectivity [41][42][43][44][45][46][47][48][49][50][51][52], while machine learning has been under-utilised. Machine learning methods are described either as discriminative or generative (Figure 1.3) [53,54] and may be applied to classification, e.g.…”

“…Previous modelling studies predicting the preferred enantiomer and the degree of enantioselectivity have primarily used quantitative structure-activity relationship (QSAR) or molecular dynamics methods [41][42][43][44][45][46][47][48][49][50][51][52]. Such methods often require high-resolution protein structures, either derived from crystallography experiments or homology modelling, and need to explicitly model environmental variables that may influence enzyme enantioselectivity, e.g.…”

“…The use of enzymes to produce enantiopure products (from a racemic substrate) is highly desirable to industry because they offer the potential to improve economics of chemical synthesis as well as lower environmental impact. For these reasons, methods to predict and design enzymes with improved stereoselectivity (both enantio-and diastereo-) has been an area of intense research [272,273,46,47,274,48,74,275,268,52,75,28].…”