REGULATORY AND STRUCTURAL ANALYSIS OF UMP SYNTHASE

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UMP synthase is a multifunctional protein containing both phosphoribosyltransferase (OPRTase) and orotidine-5'-monophosphate decarboxylase (ODCase). These two enzymes are the last of a sequence of six enzymes required for de novo biosynthesis of UMP, a precursor of all other pyrimidine nucleotides. UMP synthase is elevated in rapidly growing tissues and cells such as those of tumor origin and has been a target enzyme for chemotherapy. In contrast to tumor tissue, the genetic disease, orotic aciduria, has been described and directly results from a defective UMP synthase lacking either both OPRTase and ODCase (Type I) or ODCase (Type II). Chronic uridine therapy overcomes the manifestations of the disease. A cDNA having the coding sequence for the ODCase domain of UMP synthase, has been isolated and expressed by use of appropriate vectors in yeast and E. coli mutants lacking this protein. We will continue to try to isolate the complete cDNA for UMP synthase and/or a cDNA for the OPRTase domain which lies 5' from the coding sequence of the ODCase domain of pMEJ. The cDNA for UMP synthase will be sequenced to allow the amino acid sequence of the protein to be determined. Use of appropriate expression vectors should allow expression of UMP synthase in appropriate E. coli or yeast mutants. The coding region of the UMP synthase cDNA will be altered. These novel cDNAs will be used to program OPRTase- deficient and ODCase deficient E. coli to produce new truncated proteins which complement only one deficiency. These DNAs will be analyzed by restriction mapping and DNA sequencing to determine which amino acids of the entire amino acid sequence constitute the individual OPRTase and ODCase catalytic domains. Few direct studies have been done to identify amino acid side chains required for both enzyme activities of UMP synthase. Protein modification studies using yeast ODCase (which can be isolated in large quantities) will initiate such studies as well as a search for conditions necessary to crystallize this protein for X-ray studies. These protein studies should aid in identifying sites in the ODCase domain of UMP synthase where site-specific mutations could produce significance modification. When the cDNA for UMP synthase becomes available such studies can be extended to the bifunctional protein and the OPRTase domain.