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.

Public Health Relevance

The primary objective for the research described in this proposal is directed towards the determination of the chemical reaction mechanism and three-dimensional structure of a novel bacterial phosphotriesterase that is capable of hydrolyzing and detoxifying organophosphate nerve agents, plasticizers, flame retardants, and household pesticides. This enzyme will be used as a template for the creation of novel enzymes with enhanced catalytic properties for the detection, destruction, and detoxification of organophosphate nerve agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM116894-02
Application #
9407189
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Barski, Oleg
Project Start
2017-01-01
Project End
2020-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Texas A&M University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
020271826
City
College Station
State
TX
Country
United States
Zip Code
77845