Principal Investigator: JOHN GERLT
Affiliation: University of Illinois
Country: USA
Abstract: Structure-based explanations for the catalytic efficiencies and substrate specificities of enzymes are elusive. The goals of this project include 1) providing a structure-based explanation for the extraordinary catalytic efficiency of orotidine 5'-monophosphate decarboxylase (OMPDC) that catalyzes the final step in the pathway for pyrimidine nucleotide biosynthesis, and 2) characterizing the substrate specificity and mechanistic diversity in the D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) superfamily. Specific Aim 1 focuses on the roles of active site residues in both ground state destabilization (GSD) and transition state stabilization (TSS) in the OMPDC-catalyzed reaction. We have established that the reaction involves an intermediate, and we now want to understand the mechanisms by which the intermediate is generated and stabilized. We will examine the roles of specific amino acid residues in stabilizing negative charge that is delocalized in the pyrimidine ring of the intermediate (TSS) as well as those putatively involved in destabilizing the substrate carboxylate group (GSD). We will collaborate with Drs. Arthur Palmer, Columbia, for NMR studies of structure and dynamics and Dr. Jiali Gao, Minnesota, for computational studies. Specific Aim 2 focuses on the structural mechanism by which substrate binding is coupled to catalysis in the OMPDC-catalyzed reaction. Studies using both site-directed mutagenesis and "two part" substrate analogs (phosphite anion and a truncated orotidine, Drs. John Richard and Tina Amyes, Buffalo) demonstrate that the remote phosphate group synergizes decarboxylation. We will examine the conformational changes that accompany substrate binding to establish the roles of specific residues in coupling binding to catalysis. Specific Aim 3 is focused on characterizing mechanistic diversity in the RuBisCO superfamily. The structures suggest that the uncharacterized members will be excellent candidates for functional prediction using an integrated experimental and computational approach that successfully identified novel reactions in the amidohydrolase and enolase superfamilies;we will collaborate with Dr. Matthew P. Jacobson, UCSF, for both homology modeling and in silico library docking. As new functions are discovered, the mechanisms will be characterized so that the structural bases of functional and mechanistic diversity can be described. These studies will contribute to 1) delineating structural strategies for the design of inhibitors of OMPDC (beyond the scope of the current project);and 2) enhancing integrated structure-function-computation approaches for assigning the functions of uncharacterized proteins discovered in genome projects. PUBLIC HEALTH RELEVANCE: This project is focused on the important biomedical problem of exploiting genomic information to both assign functions to proteins discovered in genome sequencing projects and, also, to establish the structural bases for the biological functions. If new targets and experimental approaches are to be devised for small molecule intervention (drugs), the roles of all proteins involved in an organism's molecular, cellular, and organismal functions must be known. This project uses orotidine 5'-monophosphate decarboxylase (OMPDC), an essential enzyme in the biosynthesis of RNA and DNA, and homologues of D-ribulose 1,5-bisphosphate carbxoylase/oxygenase (RuBisCO), the ubiquitious enzyme that fixes CO2, to develop new knowledge about the structural basis for biological function. Our studies of OMPDC-catalyzed reaction will enable new strategies for the design of inhibitors for use as antibiotics;our studies of the RuBisCO superfamily will enhance the development of new approaches for assigning the functions of uncharacterized proteins discovered in genome projects.
Funding Period: ----------------2003 - ---------------2012-
more information: NIH RePORT