This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Carboxylesterases (CEs) are promiscuous metabolizing enzymes that catalyze the hydrolysis of esterified xenobiotics and endobiotics. The pyrethroid insecticides are ester-containing environmental pollutants widely found in the southeastern U.S. due to their extensive use in the agricultural industry. When humans are exposed to pyrethroids, CEs are the primary enzymes in the liver responsible for detoxifying and clearing these chemicals from the body. However, the mechanism by which CEs recognize pyrethroids and catalyze their hydrolysis is not completely characterized. For instance, permethrin is a widely used pyrethroid used as a mixture of cis- and trans-isomers. These isomers are stereoselectively metabolized by CE enzymes, for example the trans-isomer is cleaved markedly faster than the cis-isomer. What accounts for this selectivity? This is one of the primary questions being addressed by this pilot project. Recently, a carboxylesterase found in Bacillus subtilis (termed pnb CE) was shown to possess 81% amino acid sequence identity and a high degree of structural homology with a mammalian CE, rabbit liver carboxylesterase (Wierdl et al., 2004). Importantly, pnb CE did not need to be glycosylated to be enzymatically active and expression of large amounts of this enzyme can be done rapidly and conveniently in E. coli. Thus, sequence variants in pnb CE cDNA can be quickly constructed yielding enzymes with site-specific amino acid modifications at key locations involved in substrate recognition and catalysis. The objective of this pilot project will be to use wild type and pnb CE mutants to study the catalytic mechanism of pyrethroid hydrolysis. The central hypothesis for the research is: Mutant carboxylesterases have altered substrate binding affinities and/or catalytic steps compared with wild type enzyme, and these enzymes will provide insight into the catalytic mechanism of carboxylesterases.
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