Year: 2025
Type: article
Abstract: Toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1 was investigated as a drug-metabolizing enzyme for the first time and was found to metabolize chlorzoxazone and resveratrol to form human metabolites 6-chlorzoxazone (0.045 ± 0.016 nmol/hr/mg protein) and piceatannol (0.014 ± 0.009 nmol/hr/mg protein), respectively, though at low rates. ToMO also forms 2-acetamidophenol (2-AAP, 27%), 3-AAP (42%), and 4-AAP (31%) from acetanilide at 3.6 ± 0.3 nmol/hr/mg protein. Multiple-site saturation mutagenesis at positions I100/E103/A107 of the alpha-subunit along with site-directed mutagenesis approaches were used to isolate thirty-seven different ToMO variants with enhanced activities and/or fine-tuned specificities. Specifically, variant I100V/E103T was identified with 2.1- and 49-fold higher activities towards acetanilide and chlorzoxazone, respectively, compared to native ToMO. Variant I100V/E103T also had the regiospecificity of acetanilide change from 31% to 100% 4-AAP, mimicking human liver enzyme behavior. In addition, several variants showed up to 3.7-, 1.6-, and 3.2-fold improved selectivity for 2-, 3-, and 4-AAP formation, respectively. For resveratrol, variant I100T/E103L was a better catalyst than native ToMO, exhibiting 34-fold higher activity. The results presented here demonstrate the potential of nonhuman ToMO variants in drug metabolism and contribute to the list of research on probing this promising enzyme.
Source: Biotechnology and Bioengineering
DOI: 10.1002/bit.70117
Authors: Noella Younan, Felice A. Dacpano, Eli Frazer, Angeline Dauz, Areli Tlatelpa, Gönül Vardar‐Schara
Institution: California State University, Stanislaus
Language: English
Subfield: Pharmacology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4416908963