Enzymatic Hydrolysis of Organophosphate Esters Grant uri icon

abstract

  • The primary objective for the research described in this proposal is the elucidation of thechemical reaction mechanism and three-dimensional structure of a novel phosphotriesterase recentlyidentified from the bacterium Spingobium sp. strain TCM1 (Sb-PTE). The high toxicity of manyorganophosphate triesters has been exploited as the active component in many commercialagricultural and household insecticides and as ultra-potent chemical warfare agents. Other, less toxic,organophosphate compounds have been widely utilized as flame retardants, plasticizers, and asprodrugs 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 fromPseudomonas diminuta (Pd-PTE) or any other enzyme identified to date. We propose to utilize thisenzyme as a template for the design and creation of new biological catalysts that can be exploited forthe detection, destruction, and detoxification of toxic organophosphate nerve agents that are currentlybeing used as agricultural and household insecticides, plasticizers, and chemical warfare agents. Thechemical mechanism for this enzyme will be elucidated by determining the stereochemical course ofthe reaction with chiral substrates and by monitoring the fate of 18-oxygen labels in the substrate andenzyme. 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. Inpreliminary experiments we have succeeded in the crystallization of Sb-PTE and determination of itsthree dimensional structure by X-ray diffraction methods. Further structures will be pursued in afocused 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 withunique substrate profiles. Rational and combinatorial libraries of mutant enzymes will be constructedand those variants with enhanced catalytic proficiency for the hydrolysis of toxic organophosphateswill be identified through unique screening and selection procedures. The changes in the amino acidsequence of the Sb-PTE mutants will be correlated with enhancements in the catalytic properties andalterations in the structure within the active site.

date/time interval

  • 2017 - 2020