Nearly all bacteria require iron to grow. Understanding the main processes by which bacteria gain iron provides valuable insight into how to disrupt the iron uptake process, and potential new strategies to limit growth of infectious microbes. The focus of this proposal is the biosynthesis of siderophores. Siderophores are natural materials produced by bacteria that bind iron very tightly, and facilitate iron uptake by the bacterium. The goal of the proposal is to figure out how bacteria make these compounds stepwise from simple building blocks. The research plan aims to understand three of these steps for a specific siderophore called pyoverdin. Portions of this proposal are structured such that undergraduate students will obtain meaningful research experiences to inspire careers in research. This proposal also includes a project to be conducted by a graduate student, providing training and mentoring of future scientists at the interface of chemistry and biology. Ongoing programs at the University of Kansas will aid in the active recruitment of under-represented minorities to undergraduate research positions. The PI is also an active participant in mentoring and recruiting students of diversity to graduate programs at the University of Kansas, and is involved in community outreach to inspire an interest in science in even the youngest (preschool) students.
This award to Dr. Audrey Lamb of the University of Kansas by the Chemistry of Life Processes Program in the Chemistry Division provides funding to investigate the enzymes of hydroxamate siderophore biosynthesis. The first enzyme under study, the ornithine hydroxylase, is a class B flavoprotein monooxygenase with interesting properties that make it an excellent system for crystallographic visualization of the flavin-oxygen intermediates of the catalytic cycle. The second enzyme to be studied is the hydroxyornithine formyl-transferase, a putative 10-formyl-tetrahydrofolate-dependent glycinamide ribonucleotide transformylase-like enzyme, for which we will provide the first biochemical and structural characterizations for this enzyme class. Finally, in a project driven by undergraduate researchers, we will study the substrate specificity of the adenylation and condensation domains of the nonribosomal peptide synthetase PvdJ. This two module NRPS incorporates the formyl-hydroxyornithine generated by the first two enzymes under study, as well as a lysine. The experimental approaches include steady state and transient state kinetics, and x-ray crystallography. The outcomes of this project will significa ntly advance the understanding of the structure-function relationships required for catalysis. Furthermore, this proposal is designed to address our gaps in knowledge regarding flavin chemistry, N-formylation chemistry, and the formation of bioactive peptides in these three enzyme classes.
