Determining the Structure of NRPS‐Independent Siderophore (NIS) Synthetase DesD Using X‐ray Crystallography

Abstract

A lack of iron leads to secretion of siderophores (small molecule metal chelators) by bacteria. Siderophores have high affinity for iron outcompeting host chelators, and making them virulence factors in infection. Some siderophores (of particularly virulent pathogens) are assembled via an NRPS‐independent siderophore (NIS) pathway. Understanding the structure of NIS siderophore biosynthesis enzymes may lead to the design of specific antibiotics to fight pathogenic bacteria.The Hoffmann lab is using x‐ray crystallography to determine the protein structure of NIS synthetase DesD, from Streptomyces coelicolor. DesD makes the siderophore desferrioxamine B, requiring three iterative bonds and a macrocyclization event. DesD is one of the best biochemically characterized NIS synthetases so far, and we are solving this structure as a model for the rest. The goal of this project is to solve the x‐ray structure of DesD bound to substrate and ATP, as well as apo (empty of substrate) structures, in order to characterize the conformational changes that may occur with binding. DesD crystalizes in 20% PED 8,000, 15% glycerol, and 15% ethylene glycol conditions, and crystals diffract to 2.48 Å with good statistics. We are solving the structure using molecular replacement and are pursuing substrate‐bound crystals. This structural information may allow us to design an inhibitor that will prevent the binding of substrate in future work.