Thermal Stability of Type II Polyketide Acyl Carrier Proteins

Abstract

Engineering biosynthetic pathways in microorganisms is a compelling route to gain access to novel molecules with drug‐like properties. Polyketide synthases (PKSs) utilize acyl carrier proteins (ACPs) to convert simple acetate building blocks into structurally complex molecules, many of which are antibiotics and anticancer agents. In a process known as combinatorial synthesis, biochemists have attempted to mix‐and‐match enzymes from different PKSs to create new hybrid synthases capable of synthesizing novel polyketide natural products. However, the ACP has proven to be a roadblock in these studies, as little is known about how the ACP functions and interacts with other enzymes due to its small and dynamic nature. To further our knowledge about the structure and function of ACPs, we cloned, expressed, and characterized a subset of 14 type II PKS ACPs. Temperature‐dependent circular dichroism (CD) experiments were conducted to determine the thermostability of the ACPs in the presence or absence of divalent cations. Our results indicate that despite strong sequence similarity, ACPs display a range in thermal stability. Further, the presence of metal ions confers additional stability to the ACP, presumably by decreasing electrostatic repulsive forces. Analytical ultracentrifugation‐sedimentation velocity experiments revealed that all ACPs sediment as monomers, and that despite sharing a common ancestor with fatty acid synthases, only one of the PKS ACPs studied interacted with the E. coli fatty acid ketosynthase, FabF.Support or Funding InformationWe would like to acknowledge an NSF CAREER Award #R15GM120704 and an NIH Award #CHE‐1652424 to Louise K. Charkoudian, as well as the Beckman Scholars Program, as sources of funding for this project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.