# Enzymatic Hydrolysis of Organophosphate Esters

> **NIH NIH R01** · TEXAS A&M UNIVERSITY · 2020 · $278,226

## Abstract

The primary objective for the research described in this proposal is the elucidation of the
chemical reaction mechanism and three-dimensional structure of a novel phosphotriesterase recently
identified from the bacterium Spingobium sp. strain TCM1 (Sb-PTE). The high toxicity of many
organophosphate triesters has been exploited as the active component in many commercial
agricultural and household insecticides and as ultra-potent chemical warfare agents. Other, less toxic,
organophosphate compounds have been widely utilized as flame retardants, plasticizers, and as
prodrugs for viral infections. The catalytic properties and three-dimensional structure of the wild-
type Sb-PTE are significantly different from those exhibited by the phosphotriesterase from
Pseudomonas diminuta (Pd-PTE) or any other enzyme identified to date. We propose to utilize this
enzyme as a template for the design and creation of new biological catalysts that can be exploited for
the detection, destruction, and detoxification of toxic organophosphate nerve agents that are currently
being used as agricultural and household insecticides, plasticizers, and chemical warfare agents. The
chemical mechanism for this enzyme will be elucidated by determining the stereochemical course of
the reaction with chiral substrates and by monitoring the fate of 18-oxygen labels in the substrate and
enzyme. These experiments will be complemented by measurement of heavy atom isotope effects,
determination of the substrate/activity profile, and the identification of potent enzyme inhibitors. In
preliminary experiments we have succeeded in the crystallization of Sb-PTE and determination of its
three dimensional structure by X-ray diffraction methods. Further structures will be pursued in a
focused attempt to determine the mode of substrate binding within the active site of this enzyme.
These structures will be utilized as a guide for the design and creation of novel enzyme variants with
unique substrate profiles. Rational and combinatorial libraries of mutant enzymes will be constructed
and those variants with enhanced catalytic proficiency for the hydrolysis of toxic organophosphates
will be identified through unique screening and selection procedures. The changes in the amino acid
sequence of the Sb-PTE mutants will be correlated with enhancements in the catalytic properties and
alterations in the structure within the active site.

## Key facts

- **NIH application ID:** 9838230
- **Project number:** 5R01GM116894-04
- **Recipient organization:** TEXAS A&M UNIVERSITY
- **Principal Investigator:** Frank M. Raushel
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $278,226
- **Award type:** 5
- **Project period:** 2017-01-01 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9838230

## Citation

> US National Institutes of Health, RePORTER application 9838230, Enzymatic Hydrolysis of Organophosphate Esters (5R01GM116894-04). Retrieved via AI Analytics 2026-06-02 from https://api.ai-analytics.org/grant/nih/9838230. Licensed CC0.

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