UMP SYNTHASE AND THE MOLECULAR BASIS OF OROTIC ACIDURIA

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UMP synthase is a bifunctional protein catalyzing the final two steps in the de novo biosynthesis of uridine 5' monophosphate (UMP). The two activities, orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC), catalyze the conversion of orotic acid to orotidine=5'-monophosphate (OMP) and OMP to UMP. As determined from the nucleotide sequence of the cDNA, UMP synthase is composed of 480 amino acids and has a molecular weight of 52,119. Hereditary orotic aciduria is a autosomal recessive disease associated with a severe deficiency of both activities of UMP synthase (Type I), or deficiency of only the ODC activity, (Type II). Orotic aciduria is the only specific disorder of de novo pyrimidine nucleotide biosynthesis described in humans. One of the primary objectives of this project is to identify the mutations in the structure of the UMP synthase protein associated with orotic aciduria. The sequence of the UMP synthase cDNA from orotic aciduria cells will be compared to the normal sequence. Sequence alterations identified in the mutant will be confirmed by various methods, including synthesis of the mutant protein from cDNA inserted into expression vectors, hybridization of allele specific oligonucleotides, or altered restriction site analysis. Another primary objective is to understand how the catalytic domains of UMP synthetase interact to form the normal bifunctional protein. In prokaryotes and lower eukaryotes, the two activities of UMP synthase reside in separate proteins. In mammals the two catalytic domains are joined by a linker or connector sequence. By changing the length and amino acid composition of the linker peptide we can determine the structural requirements for connecting the two catalytic domains. With the use of expression vectors we can also produce eukaryotic cells that synthesize separate proteins with OPRT and ODC activity or a single protein with the arrangement of the domains reversed. The stability and kinetic properties of the altered proteins will be compared to that of the normal bifunctional protein. Analysis of the defects in UMP synthase associated with orotic aciduria will increase our understanding of genetic defects and their effects on protein structure and activity. The characterization of the interaction of catalytic domains will provide information regarding enzyme evolution and cooperation, gone fusion, and substrate channeling.