Nucleoside monophosphate synthesis provides the components of DNA, RNA, ATP and NAD and requires the action of phosphoribosyltransferases (PRTases) to transfer the ribose 5-phosphate group from its donor PRPP to specific heterocyclic bases. The PRTases are the site of several life-threatening hereditary disorders, and are legitimate targets for anti-parasitic, anti-microbial and anti-tumor agents. This consortium seeks to understand the structure and function of the PRTase enzyme group. Orotate PRTase, hypoxanthine-guanine PRTase and quinolinate PRTase have been crystallized and their three dimensional structures determined. The project seeks to reveal the structural and chemical details of enzymic catalysis through studies of OPRTase and QAPRTase. Using chemical quench techniques, the OPRTase kinetic mechanism will be detailed to permit understanding of newly discovered negative cooperativity of binding and catalysis. The OPRTase active site borders the subunit interface, and requires a mobile protein structural element (loop) contributed by the adjacent subunit. Structural interactions at the subunit interface may be responsible for cooperativity. The contribution to active site chemistry of the flexible element will be determined using point mutants and partial loop deletants and by determining the structure of enzyme complexed with novel synthetic bi-substrate analogue inhibitors. The catalytic function of a pair of carboxylate residues, common to Class 1 PRTases, will be determined. QAPRTase represents a newly discovered evolutionary origin and novel architecture (the Class 2 PRTase fold) for the PRTase enzyme group. The X-ray structure of QAPRTase will be refined and the catalytic role of active site residues structurally analogous to those in OPRTase will be determined.
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