Structural and Functional Large Substrate Binding in Iterative Non‐ribosomal Peptide Synthesis Independent Synthesis (NIS) Enzymes

Abstract

Prokaryotes scavenge iron using small molecule chelators called siderophores, made through a novel chemistry of peptide bond formation. One type of siderophore synthesis, 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 multiple bonds may be catalyzed on the same substrate (iterative.) Prior work in the field has identified a novel protein fold as well as the unique peptide bond chemistry, but no structures exist of an iterative protein. The iterative activity is correlated with broad substrate specificity, but this has never been delineated nor quantified. In fact, no iterative enzyme kinetic parameters have been satisfactorily described. We propose basic science to characterize the structure, function, and elasticity of the under‐described iterative proteins in this novel family of NIS sythetases. To do this, we will utilize the following key methodologies: (1) site‐directed mutagenesis to probe important residues; (2) isothermal titration calorimetry (ITC) to explore the thermodynamics of substrate and cofactor binding; (3) Kinetic assays to quantify and compare iterative vs. single bond formation; and (4) X‐ray crystallography to probe the structural changes of iterative substrate binding. The results of our work will be used as a foundation for future mechanism and inhibitor studies, an effort that could lead to a new class of antibiotics.Support or Funding InformationThis work was supported in part by a grant from the National Science Foundation (MCB‐1716986) and by California Lutheran University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.