Browsing by Author "Raushel, Frank"

Now showing 1 - 2 of 2

Enzymatic Hydrolysis of Organophosphate Esters
Chemistry; TAMU; https://hdl.handle.net/20.500.14641/498; National Institutes of Health
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.
Novel Biochemical Pathways for the Metabolism of Carbohydrates in the Human gut Micriobiome
Chemistry; TAMU; https://hdl.handle.net/20.500.14641/498; DHHS-NIH-National Institute of General Medical Science
The primary focus of this research proposal will be the identification, discovery, and elucidation of novel biochemical pathways for the metabolism of complex carbohydrates in the human gut microbiome. Currently, more than one thousand different bacterial species have been identified in the human intestinal tract and the total number of genes contained within these bacteria exceeds the number of human genes by more than two orders of magnitude. Moreover, it has been demonstrated that the composition of the human gut microbiome and the associated metabolic diversity contained within these bacteria contribute significantly to the maintenance of human health and physiology. Unfortunately, a significant fraction of the enzymes and corresponding metabolic pathways contained within the bacteria found in the human gut have an uncertain, unknown, or incorrect functional annotation. This uncertainty suggests that a substantial fraction of the metabolic potential contained within the human gut microbiome remains to be properly characterized. Our proposed experimental approach for the discovery and elucidation of novel metabolic pathways for the metabolism of complex carbohydrates will involve the concerted and synergistic utilization of bioinformatics, computational biology, three-dimensional protein structure determination, metabolomics and physical screening of focused compound libraries. The determination of the substrate and reaction diversity contained within the newly discovered enzyme-catalyzed reactions will provide unique insights into the molecular mechanisms for the evolution and development of novel enzymatic activities and will provide potential targets for therapeutic intervention.