Synthesis and Validation of Acetyl- and Malonyl-CoA Analogs for the Study of Substrate Specificity and Mechanism of Acyltransferases

Acyltransferases (AT) are seen in a wide variety of metabolic functions. These include enzymes in polyketide synthase pathways (PKSs), fatty acid biosynthesis, energy production, and secondary metabolism. The products from these enzymes often include pharmaceutically important natural products. ATs can transfer a variety of metabolites, such as acetyl, malonyl, succinyl, and larger chain fatty acyl groups using Coenzyme A (CoA). The reactivity of the thioester sulfur allows for transfer of these metabolites to secondary metabolites or enzymes. One such example of an AT is type III chloramphenicol acetyltransferase (CATIII) from E. coli. CATIII attaches the acetyl moiety of acetyl-CoA to a hydroxyl group on the antibiotic chloramphenicol, thus conferring chloramphenicol resistance to bacterial cells via O-acetylation. Previously, the structure of CATIII had been solved with its first substrate, chloramphenicol, bound. However, the interaction between CATIII and its second substrate, acetyl-CoA, is not understood very well. Through x-ray crystallography and the synthesis of analogs of acetyl-CoA, this project aims to elucidate the interaction between CATIII and both of its substrates (chloramphenicol + acetyl-CoA). This will validate the usefulness of a wide variety of acyl-CoA analogs for use in other acyltransferases.