Engineering a BsBDHA substrate-binding pocket entrance for the improvement in catalytic performance toward (R)-phenyl-1,2-ethanediol based on the computer-aided design

Oxidation reactions of vicinal diols using (2,3)-butanediol dehydrogenases hold promise for producing & alpha;-hydroxy ketones or in cascade biocatalytic processes for chiral amino alcohols. However, their application is restricted by low specific activities and catalytic efficiencies. In this study, to enhance the catalytic activity toward (R)-phenyl1,2-ethanediol (PED), a Bacillus subtilis (2,3)-butanediol dehydrogenase (BsBDHA) was engineered by reshaping its substrate-binding pocket (SBP) entrance based on computer-aided design. Firstly, based on molecular docking simulation, seven residues of BsBDHA were identified and subjected to leucine scanning mutagenesis. Two E. coli transformants, E. coli/bsbdhaI49L and /bsbdhaV266L, were confirmed with oxidation activities of 0.85 and 0.87 U/g wet cell, respectively, being 1.20- and 1.23-fold that of E. coli/bsbdha. Secondly, both Ile49 and Val266 were subjected to partial site-saturation and combinatorial mutagenesis. One double-site mutant, BsBDHAI49L/V266L, was obtained and its specific activity and kcat/Km toward (R)-PED increased to 1.79 U/mg and 0.39 mM-1 s � 1, respectively. These values were 3.3- and 4.9-fold those of BsBDHA. Finally, the analysis of molecular mechanism for BsBDHAI49L/V266L with enhanced catalytic efficiency was performed by molecular dynamics simulation. This study is the first report on the improvement of catalytic performance of BsBDHA toward (R)-PED and provides useful reference data for the further studies.

Automated summary

In this study, a Bacillus subtilis (2,3)-butanediol dehydrogenase (BsBDHA) was engineered to enhance its catalytic activity toward (R)-phenyl1,2-ethanediol (PED) by reshaping its substrate-binding pocket (SBP) entrance. Mutagenesis experiments were performed to identify key residues and a double-site mutant, BsBDHAI49L/V266L, was obtained with significantly improved specific activity and catalytic efficiency. Molecular dynamics simulation was used to analyze the molecular mechanism of enhanced catalytic efficiency. This study provides valuable reference data for further studies.