The biocatalyst toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1 belongs to the great family of bacterial multicomponent monooxygenases and has been shown to have a potential for biotechnological and environmental applications. Acetanilide, which was marketed under the name Antifebrin, has been used as a model substrate in numerous drug metabolizing human P450 monooxygenase studies. In this study, metabolism of acetanilide was examined with native and engineered ToMOs using molecular modeling and site directed mutagenesis. A molecular docking-based approach was first utilized for identifying the best amino acid substitution(s) at selected active site residues on the basis of acetanilide’s predicted binding affinities as well as its orientation inside the binding site. ToMO site and combinatorial saturation mutagenesis libraries were generated using the PyMOL software and virtual screening was performed towards acetanilide using the AutoDock Vina software. A total of two hit ToMO variants with a better docking results compared to wild-type ToMO were chosen for site directed mutagenesis studies followed by enzymatic activity assay to determine the acetanilide oxidation activity levels and regiospecificities. We found that regiospecificity on acetanilide has been improved by up to 3.0-fold relative to the wild type and the predicted key mutations perturbing ToMO’s active site facilitated human P450-like hydroxylation of acetanilide. This study confirms the advantages of protein engineering approaches, shows the potential of ToMO and its variants in drug metabolism applications, and adds to the list of research on probing this remarkable enzyme.

Analyzing the potential of toluene o-xylene monooxygenase in acetanilide metabolism by molecular modeling and site directed mutagenesis

ACS Spring 2022

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