Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Populus tomentosa were cloned and studied and active sites in CCRs were further identified based on sequence divergence, molecular simulation, and site-directed mutants. Cinnamoyl-coenzyme A (CoA) reductase (CCR) is the first committed gene in the lignin-specific pathway and plays a role in the lignin biosynthesis pathway. In this study, we cloned 11 genes encoding CCR or CCR-like proteins in Populus tomentosa. An enzymatic assay of the purified recombinant P. tomentosa (Pto) CCR and PtoCCR-like proteins indicated that only PtoCCR1 and PtoCCR7 had detectable activities toward hydroxycinnamoyl-CoA esters. PtoCCR1 exhibited specificity for feruloyl-CoA, with no detectable activity for any other hydroxycinnamoyl-CoA esters. However, PtoCCR7 catalyzed p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA with a preference for feruloyl-CoA. Site-directed mutations of selected amino acids divergent between PtoCCR1 and 7, combined with modeling and docking, showed that A132 in CCR7 combined with the catalytic triad might comprise the catalytic center. In CCR7, L192, F155, and H208 were identified as the substrate-binding sites, and site-directed mutations of these amino acids showed obvious changes in catalytic efficiency with respect to both feruloyl-CoA and sinapoyl-CoA. Mutant F155Y exhibited greater catalytic efficiency for sinapoyl-CoA compared with that of wild-type PtoCCR7. Finally, recent genome duplication events provided the foundation for CCR divergence. This study further identified the active sites in CCRs and the evolutionary process of CCRs in terrestrial plants.

Chiral oxazolidinones are a class of important heterocyclic compounds in pharmaceutical chemistry due to their biological activity. Halohydrin dehalogenase-catalyzed epoxide ring-opening reaction with cyanate offers an attractive approach to the synthesis of chiral oxazolidinones, but the α/β-regioselectivity and stereoselectivity are still un-addressed issues. In this study, a unique halohydrin dehalogenase (AbHheG) was found to have high activity and α/β-regioselectivity toward the ring opening of racemic styrene oxide with cyanate but with poor stereoselectivity (E < 3). By reshaping the substrate-binding site of AbHheG, a variant Y15M/N182S was obtained with excellent α/β-regioselectivity and stereoselectivity. The variant showed E > 200 for 9 of the 13 tested styrene oxides. Since (R)-4-aryl-2-oxazolidinones were easily separated from (R)-styrene oxides, both (R)- and (S)-4-aryl-2-oxazolidinones could be readily prepared. Crystallographic and enzyme–substrate docking analysis showed that flipping of the substrate in the binding site resulted in the R-configuration substrate being away from the catalytic triad in the mutant, which was responsible for the enhanced α/β-regioselectivity and stereoselectivity. This work has demonstrated that halohydrin dehalogenase is a useful biocatalyst for the synthesis of both enantiomers of 4-aryl-2-oxazolidinones from readily available racemic styrene oxides. The structural and computational studies provide a guidance for further engineering of halohydrin dehalogenases to control the α/β-regioselectivity and stereoselectivity for the efficient synthesis of the desired optically pure 4- or 5-substituted 2-oxazolidinones.

Regio- and stereoselective reductive amination of diketones offers an attractive method to access chiral β-amino ketones with multiple stereocenters which are unique scaffolds and building blocks for bioactive molecules, but it is still a great challenge in organic chemistry. In this study, mutant amine dehydrogenases (LsAmDHs) were created by directed evolution of a l-phenylalanine dehydrogenase from Lysinibacillus sphaericus to catalyze the desymmetric reductive amination of 2,2-disubstituted-1,3-cyclopentadiones. Using these beneficial LsAmDHs, the corresponding (2R, 3R)-β-amino ketones with an all-carbon quaternary stereocenter were prepared with up to 99% de and ee and up to 84% isolated yields. A cyclopenta[b]hydroquinoline derivative was obtained from 2-methyl-2-(2′-bromobenzyl)-1,3-cyclopentadione or 2-methyl-2-(2′-chlorobenzyl)-1,3-cyclopentadione via sequential enzymatic desymmetric reductive amination and an intramolecular Buchwald–Hartwig cross coupling reaction. Molecular docking and dynamics simulations provided some insights into the roles of key mutations on the improved activity and excellent stereoselectivity toward these un-native substrates. This study not only developed biocatalysts to realize the unprecedented desymmetric reductive amination of 2,2-disubstituted-1,3-cyclopentadiones to the single stereoisomer of the corresponding β-amino ketones but also suggested that engineering of amine dehydrogenase provides a useful tool to address the challenges in asymmetric reductive amination of diketones.