Structure-guided stereoselectivity inversion of a short-chain dehydrogenase/reductase towards halogenated acetophenones

Abstract

The structure-guided rational design of an NADH-dependent short-chain dehydrogenase/reductase (SDR) reversed the stereoselectivity towards halogenated acetophenones from Prelog to anti-Prelog. The enzyme-substrate interactions involving an aromatic ring and a halogen atom were proven to play critical roles in determining the stereoselectivity of these ketone reductions besides the steric repulsion.

Single‐Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward β‐Ketoesters with Enhanced Activity

“…Consequently, β‐ketoesters 1 a – 12 a adopted typical antiPrelog‐preferred conformations, namely the nicotinamide ring of the coenzyme NADH was located on the Si ‐face of the substrates, leading to the formation of the corresponding antiPrelog alcohols. These results are in good agreement with the previous studies that the enzyme stereoselectivity could be changed by the adjustment of the relative size of the substrate‐binding pocket from different sides [13a, b, d–f] . The replacement of the bulky residue by a smaller one carved out a bigger space for the accommodation of the large group of β‐ketoesters and reversed the enzyme stereoselectivity from Prelog to antiPrelog.…”

“…Unfortunately, the trade‐off between stereoselectivity and activity is often difficult to avoid in the evolution of enzymes. For example, a significantly decreased activity was observed when the stereoselectivity of a short‐chain dehydrogenase/reductase toward halogenated acetophenones was reversed [13d] . A similar trade‐off between enantioselectivity and activity was reported for engineering of an esterase from Rhodobacter sphaeroides for the resolution of racemic methyl mandelate [14] .…”

Reconstruction of the Catalytic Pocket and Enzyme–Substrate Interactions To Enhance the Catalytic Efficiency of a Short‐Chain Dehydrogenase/Reductase

“…To identify the targets for protein engineering, substrate docking was performed by molecular dynamics simulation on the basis of the homology model of EbSDR8, and the complex model of wildtype EbSDR8 and substrate 1 d was employed as a representative model (Figure ). In the model of the enzyme–substrate complex, the substrate molecule is bound in the catalytic cavity formed by the G94, H145, S153, Y188, and L193 residues.…”

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

“…Yu and coworkers reported an inversion in stereopreference of Pseudomonas putida ADH from Prelog to anti ‐Prelog in the asymmetric reduction of halogenated acetophenones. [77] Their study was guided by structural comparison with Empedobacter brevis ADH, an anti ‐Prelog ADH, which led to identification of M85, L136, W182, and M187 as amino acids that could control the Prelog stereorecognition in P. putida ADH. Single and double point mutations at these sites resulted in P. putida ADH mutants that exhibit anti ‐Prelog stereopreference.…”