Biocatalytic characterization of an alcohol dehydrogenase variant deduced from Lactobacillus kefir in asymmetric hydrogen transfer

Abstract

Hydrogen transfer biocatalysts to prepare optically pure alcohols are in need, especially when it comes to sterically demanding ketones, whereof the bioreduced products are either essential precursors of pharmaceutically relevant compounds or constitute APIs themselves. In this study, we report on the biocatalytic potential of an anti-Prelog (R)-specific Lactobacillus kefir ADH variant (Lk-ADH-E145F-F147L-Y190C, named Lk-ADH Prince) employed as E. coli/ADH whole-cell biocatalyst and its characterization for stereoselective reduction of prochiral carbonyl substrates. Key enzymatic reaction parameters, including the reaction medium, evaluation of cofactor-dependency, organic co-solvent tolerance, and substrate loading, were determined employing the drug pentoxifylline as a model prochiral ketone. Furthermore, to tap the substrate scope of Lk-ADH Prince in hydrogen transfer reactions, a broad range of 34 carbonylic derivatives was screened. Our data demonstrate that E. coli/Lk-ADH Prince exhibits activity toward a variety of structurally different ketones, furnishing optically active alcohol products at the high conversion of 65-99.9% and in moderate-to-high isolated yields (38-91%) with excellent anti-Prelog (R)-stereoselectivity (up to >99% ee) at substrate concentrations up to 100 mM.

Fig. 1. The Prelog’s rule for predicting the stereochemical outcome of ADH-catalyzed asymmetric reduction of prochiral carbonyl compounds.

The dashed red line means below the plane.

Fig. 2. Optimization of the reaction conditions for the E. coli/Lk-ADH Prince-catalyzed asymmetric bioreduction of the model pentoxifylline (1a).

a Effect of the type of an aqueous reaction medium and addition of the external NAD(P)H cofactors. b Effect of the substrate concentration. c Effect of the propan-2-ol (2-PrOH) concentration. d Reaction scale-up and impact of the amount of E. coli/Lk-ADH Prince cells. All the reactions were conducted for 24 h at 30 °C, 250 rpm (orbital shaker). The “IPA” abbreviation states for propan-2-ol.

Fig. 3. Substrate scope for E. coli/Lk-ADH Prince-catalyzed bioreduction of carbonyl compounds 1a–ah.

With blue color were highlighted products obtained with high-to-excellent enantiopurity (>80% ee).

Fig. 4. Optimization of the reaction conditions for the E. coli/Lk-ADH Prince-catalyzed asymmetric bioreduction of 1-(biphenyl-4-yl)ethanone (1g) and 1,2-diphenylethanone (1af).

a and b Effect of the organic co-solvent (20% v/v) on the conversion of the E. coli/Lk-ADH Prince-catalyzed reduction of 1g (a) or 1af (b) in the presence of 2-propanol (10% v/v). c and d Effect of the concentration of substrate 1g (c) or 1af (d) in the presence of DMSO (20% v/v) and 2-propanol (10% v/v) as co-solvents. All the reactions were conducted with 1g or 1af (10 mM final conc. in 0.5 mL total volume), lyophilized E. coli/Lk-ADH Prince (10 mg), and 1.0 mM MgCl₂ for 24 h at 30 °C, 250 rpm (orbital shaker).

Fig. 5. Chemical structures of tenofovir (TFV), tenofovir 5′-disoproxil fumarate (TDF), tenofovir 5′-alafenamide (TAF), and their key precursor – (2R)-1-(6-chloro-9H-purin-9-yl)propan-2-ol [(R)-2w].

With blue color were highlighted common core structure.

Fig. 6. Binding mode of 1-(6-chloro-9H-purin-9-yl)propan-2-one (1w) with Lk-ADH Prince prepared from Lk-ADH (PDB code: 4RF2), with close contacts to amino acid residues and NADPH cofactor located in the active site.

The docked ligand 1w and the cofactor are shown as sticks representation, where 1w is white, and NADPH is violet. The overall receptor structure is shown as a semi-transparent light-blue cartoon (a) or sphere diagram (c), respectively. The most significant amino acid (AA) residues contributing to the stabilization of the ketone 1w in the complex with Lk-ADH Prince are shown in light-blue (for conserved AA residues) and gold (for mutated AA residues) lines representations. Nitrogen atoms are presented with blue color, oxygen atoms with red color, chlorine atoms with green color, whereas phosphorus atoms with orange color. All the hydrogens were omitted for clarity. The formation of intermolecular hydrogen bonds is represented by yellow dashed lines, whereas the plausible trajectory of the hydride transfer from NADPH to a carbon atom of the carbonyl group is shown as a red dashed line. Mutual distances between the amino acid residues and the respective ligand’s atoms are given in Ångström (b). The figures shown in panels a–c were prepared using the program PyMOL (http://www.pymol.org/). d The 2D protein-ligand interactions map was generated using the program BIOVIA Discovery Studio Visualizer 20.1.0.19295 (Dassault Systèmes Biovia Corp.; https://www.3ds.com). In this case, polar contacts between receptor-ligand including conventional hydrogen bonds (green) and π−donor hydrogen bonds (green-pastel), as well as non-polar contacts between receptor-ligand including π–σ interactions (purple/violet), π–alkyl (light pink), π–π T-shaped (pink), and π–sulfur (gold) are represented by dashed lines. Intermolecular Van der Waals forces are displayed in light-green spoked arcs.

Fig. 7. Visualization of the tunnels in wild-type Lk-ADH (panel a and b) and Lk-ADH Prince (panel c and d) represented as a set of intersecting spheres using CAVER Analyst 2.0 software.

Indicates the access pathways to buried active sites in Lk-ADH and Lk-ADH Prince, respectively. The overall enzyme structures are shown as a semi-transparent cyan (a, b) or green (c, d) cartoon, respectively. The most significant amino acid residues responsible for the formation of the tunnels and bottlenecks, as well as the NADPH cofactor, are represented by sticks. For details concerning tunnel statistics, see Supplementary Table 9 deposited in Supplementary Information.