Ni_2011_Chem.Biol.Drug.Des_78_301

The asymmetric catalysis, as the character of enzyme, attracts increasing attention from the scientific and industrial communities. In this study, the Bacillus subtilis lipase A, as a model enzyme, is studied systematically to dissect its stereoselectivity toward (rac)-ketoprofen vinyl ester using a combination scheme of molecular docking and quantum mechanical/molecular mechanical (QM/MM) analysis. In this procedure, the rational orientation of the two enantiomers of ketoprofen vinyl ester is obtained with the AutoDock performing, and then, the steric contacts between the enzyme and substrate in the docking outputs are examined visually at the atomic level with a small-probe technique. Subsequently, the binding energies of the enzyme-substrate complexes are calculated using an ONIOM (Our own N-layered Integrated Molecular Orbital + Molecular mechanics)-based QM/MM protocol. The results obtained from the theoretical studies show that the B. subtilis lipase A prefer to hydrolyze the (R )-ketoprofen vinyl ester when compared to its (S )-enantiomer, with a relatively high E (stereoselectivity) value of 31.28 charactering its enantioselectivity. Furthermore, to verify the conclusions from the computational analysis, the B. subtilis lipase A gene is cloned to overexpress the recombinant B. subtilis lipase A, and its stereoselectivity was determined. Satisfactorily, the experimental results are in well agreement with the theoretical predictions because the (R )-ketoprofen vinyl ester is found as the preferring enantiomer of the B. subtilis lipase A, with experimentally measured E value of 36.7. We therefore expect that this in silico-in vitro hybrid approach can provide a new and effective avenue to predict the catalytic activity of and to investigate the molecular mechanism of enzyme-mediated asymmetric catalysis and help in understanding the enzymatic process and in rational enzyme design.