The role of oxyanion holes in the structure and function of type III polyketide synthases (768.18)

Abstract

The objective of this study was to combine the tools of protein crystallography and enzyme kinetics to explore the structural mechanisms of type III polyketide synthases (PKSs). Type III PKSs are proteins involved in diverse metabolic processes including cell signaling, pathogen defense and the development of reproductive cells. Mechanistically, these enzymes generate metabolites through the repetitive addition of acetate units onto a starter molecule; a process that produces reactive intermediates that are ultimately cyclized into the final product. How these enzymes control their reactive intermediates and terminal cyclizations is not fully understood and is a major hindrance to protein engineering efforts. We present here our results describing the role of oxyanion holes in the mechanism of type III PKSs. Crystallographic studies of reaction analogs trapped in the active site revealed that remnants of an ancient oxyanion hole are involved in stabilizing reactive intermediates. Unexpectedly, we observed that a small molecule, metazachlor, has different conformations in the active site of functionally distinct PKSs. Enzyme kinetic studies supported our structural findings by establishing that metazachlor differentially inhibits type III PKSs based on their terminal cyclization steps. These structure‐function studies are being used as a guide for altering the terminal cyclization preferences of type III PKSs to build novel compounds.Grant Funding Source: Supported by Howard Hughes Medical Institute and National Science Foundation