RUI: Structural and Functional Substrate Binding in Iterative Non-ribosomal peptide synthesis Independent Synthesis (NIS) Enzyme DesD

California Lutheran University, Thousand Oaks CA

Investigators

Abstract

Bacteria, like humans, require iron to function optimally. For many bacteria that iron must be stolen from the host organism, often through the use of small chemicals called siderophores that bind iron. The most virulent bacteria create siderophores through a series of steps that involve one or more of an understudied family of proteins called NIS Synthetases. This project is designed to better understand this family of proteins due to their remarkable unique chemical behavior and the unusual ability to enact chemical catalysis multiple times on the same target (iterative action) as well as a never before seen structural property. The immediate goal for this project is to establish experimental methods to quantify and describe binding of targets, the structure and iterative behavior of the protein. The long-term goal of the research is to describe the mechanism of NIS synthetases in detail, to contribute understanding not only to this understudied field, but also of iterative proteins more broadly. This project will provide training and education primarily to members of underrepresented groups and first generation college students. Prokaryotes scavenge iron using small molecule chelators called siderophores, made through a novel chemistry of peptide bond formation. One type of siderophore synthesis mechanism, increasingly associated with bacterial virulence, uses Non-ribosomal peptide siderophore Independent Synthesis enzymes (NIS synthetases). Within a sub-category in this family, an additional remarkable kinetic behavior exists whereby the formation of multiple bonds may be catalyzed on the same substrate (iterative.) The iterative activity is correlated with broad substrate specificity, but this has never been delineated nor quantified. This project will characterize the structure, function, and elasticity of the iterative proteins in this novel family of NIS sythetases. Functionally important residues will be identified by site-directed mutagenesis and the use of synthetically prepared substrate analogs. Thermodynamics of ligand and cofactor binding and changes in structure with iterative substrate binding will be determined. Kinetic assays will be performed to quantify and compare iterative vs. single bond formation. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.