Structure, Mechanism, and Regulation of Quinolinate Synthase, the First Committed Step in Bacterial NAD Biosynthesis

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Intellectual Merit: Nicotinamide adenine dinucleotide (NAD) is an essential and ubiquitous metabolite known primarily for its role as a co-substrate in a multitude of biological redox reactions. Recently, however, it has received significant recognition for its role in several non-redox reactions that impact cellular signaling and chromosome remodeling. In bacteria, NAD is biosynthesized from dihydroxyacetone phosphate and L-aspartate in a reaction catalyzed by the NadA/NadB enzyme system. NadA contains an oxygen-sensitive [4Fe-4S] cluster, which is required for the protein's function; however, the exact role that the iron-sulfur (Fe/S) cluster plays in catalysis is unclear. Moreover, the activity of the protein is regulated by a dithiol/disulfide redox switch, wherein the disulfide-form of the enzyme is approximately10-fold more active than the dithiol form. This behavior is interesting, because the concentrations of NAD and its associated metabolites are significantly greater in bacteria growing under aerobic conditions than under anaerobic conditions. The major goal of this project is to use X-ray crystallographic and spectroscopic methods to elucidate in detail how the Fe/S cluster functions in catalysis. A second area of emphasis is to elucidate the structural, chemical, and electronic changes that accompany reversible disulfide-bond formation and determine how these changes affect the protein's activity and cellular concentrations of NAD and its related metabolites. Broader Impact: This project will serve as a foundation for training graduate and undergraduate students in rigorous and quantitative scientific methods. Penn State is home to a wealth of expertise in metalloenzymology, especially as it pertains to physical, kinetic, and computational methods for studying iron-containing enzymes. The PI, in collaboration with other faculty here and at other institutions, will co-organize a biannual ten day workshop to train students in various physical methods for the characterization of metalloproteins.This project is jointly supported by the Biomolecular Dynamics, Structure and Function Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes program in the Chemistry Division.