# Characterizing and engineering toluene o-xylene monooxygenase for the synthesis of common drug metabolites

> **NIH NIH SC3** · CALIFORNIA STATE UNIVERSITY-STANISLAUS · 2020 · $98,650

## Abstract

Project Summary/Abstract
As drugs are metabolized in the body, their metabolites represent new chemical entities to which humans are
exposed. Thus, investigating the toxicities and characterizing biological activities of drug metabolites is crucial
for the development of safe, effective drugs. In fact, pharmacokinetic studies are critical components of
investigational new drug applications to the US Food and Drug Administration. As a part of these studies,
however, large quantities of pure metabolites are needed to characterize them in vitro and, especially in vivo.
The chemical synthesis of drug metabolites is problematic in terms of yield, selectivity, and can be a major cost-
driver in preclinical studies. Direct enzymatic synthesis of drug metabolites is an attractive alternative. Although
P450 monooxygenase enzymes, found in human liver, have been in the spot light as drug metabolizing
biocatalysts, many challenges still remain such as low biocatalytic activity, limited drug substrate specificity and
product range. The investigation of alternate enzymes or biocatalysts may lead to a diversified metabolites
product spectra and open up new horizons for efficient metabolite production. Due to its excellent chemistry,
wide substrate range, and malleable catalytic activity, we propose to investigate the biocatalyst toluene o-xylene
monooxygenase (ToMO) of Pseudomonas sp. OX1 as a drug metabolizing enzyme. Using protein engineering
techniques, we plan to create ToMO variants with enhanced drug oxidation activity and fine-tuned specificity.
Using protein engineering, we previously created several variants of ToMO for green chemistry and
bioremediation applications. The prospect of using ToMO and its engineered variants for the synthesis of drug
metabolites presents a new and exciting approach. Our long-term goal is to improve the versatility of ToMO even
further by exploring and expanding its substrate repertoire for drug development and pharmaceutical production.
The specific aims of this project are to characterize wild-type ToMO for drug oxidation activity with 6 different
drug substrates and drug-like candidates including aniline, acetanilide, bupropion, ticlopidine, chlorphenamine,
and omeprazole (Aim 1), construct ToMO variant libraries using protein engineering and screen for further
improvements (Aim 2), and sequence, model, and characterize positive ToMO variants for drug oxidation activity
(Aim 3).

## Key facts

- **NIH application ID:** 9934818
- **Project number:** 1SC3GM136524-01
- **Recipient organization:** CALIFORNIA STATE UNIVERSITY-STANISLAUS
- **Principal Investigator:** Gonul Schara
- **Activity code:** SC3 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $98,650
- **Award type:** 1
- **Project period:** 2020-09-04 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9934818

## Citation

> US National Institutes of Health, RePORTER application 9934818, Characterizing and engineering toluene o-xylene monooxygenase for the synthesis of common drug metabolites (1SC3GM136524-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9934818. Licensed CC0.

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