Laboratory evolution of an alcohol dehydrogenase towards enantioselective reduction of difficult-to-reduce ketones

Abstract

Background: A thermostable alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) has been repurposed to perform asymmetric reduction of a series of prochiral ketones with the formation of enantio-pure secondary alcohols, which are crucial chiral synthons needed in the preparation of various pharmaceuticals. However, it is incapable of asymmetric reduction when applied to bulky ketones. Recently, mutations at two key residues A85 and I86 were shown to be crucial for reshaping the substrate binding pocket. Increased flexibility of the active site loop appears to be beneficial in the directed evolution of TbSADH towards difficult-to-reduce ketones. Methods: Using the reported mutant A85G/I86A as template, double-code saturation mutagenesis (DCSM) was applied at selected residues lining the substrate binding pocket with a 2-membered reduced amino acid alphabet. Results and conclusions: The mutant A85G/I86A was first tested for activity in the reaction of the model substrate (4-chlorophenyl)-(pyridin-2-yl)methanone, which showed a total turnover number (TTN) of 3071. In order to further improve the turnovers, a small and smart mutant library covering a set of mutations at Q101, W110, L294, and C295 was created. Eventually, a triple-mutant A85G/I86A/Q101A was identified to be a superior catalyst that gave S-selective product with 99% ee and 6555 TTN. Docking computations explain the source of enhanced activity. Some of the best variants are also excellent catalysts in the reduction of other difficult-to-reduce ketones.

Biocatalytic Oxidation of Alcohols

“…Natural enzymes have been optimized by natural evolution to serve the host organisms' purpose, which does not necessarily coincide with the needs of an organic chemist aiming at the selective oxidation of a given target molecule. Next to screening natural diversity for more suitable enzymes, protein engineering has become a very powerful tool to tailor the properties of a given enzyme such as cofactor specificity, thermo and solvent stability, (enantio)selectivity, and more [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33].…”

Alcohol Dehydrogenases with anti‐Prelog Stereopreference in Synthesis of Enantiopure Alcohols

“…Subsequently, Sun and coworkers reported A85G/I86A/Q101A Tb SADH, which was identified using combinatorial active‐site double‐code saturation mutagenesis at sites that line the substrate‐binding pocket (Q101, W110, L294, and C295) as a catalyst for asymmetric reduction of CPMK to its corresponding ( S )‐alcohol in excellent enantioselectivity. [81] …”

Biosynthesis of Chiral Amino Alcohols via an Engineered Amine Dehydrogenase in E. coli

“…The reaction mixture was proceeded at 30°C, 1,000 rpm for 24 h. The product was then detected by HPLC. TTN was defined as the molar number of the product yield divided by the catalyst concentration ( Qu et al, 2019 ).…”