Study of the kinetic and physical properties of the orotidine-5'-monophosphate decarboxylase domain from mouse UMP synthase produced in Saccharomyces cerevisiae.
In mammals, the bifunctional protein UMP synthase contains the final two enzymatic activities, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase (ODCase), for de novo biosynthesis of UMP. The plasmid pMEJ contains a cDNA for the ODCase domain of mouse Ehrlich ascites UMP synthase. The cDNA from pMEJ was joined to the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) promoter and the first four CYC1 coding nucleotides in the plasmid pODCcyc. ODCase-deficient yeast cells (HF200x1) transformed with pODCcyc expressed an active ODCase domain with a specific activity of 20 nmol/min/mg in cell extracts. The expressed ODCase domain has a lower affinity for the substrate orotidine 5'-monophosphate and the inhibitor 6-azauridine 5'-monophosphate than intact UMP synthase or an ODCase domain isolated after proteolysis of homogenous UMP synthase. Sucrose density gradient sedimentation experiments showed that the expressed ODCase domain forms a dimer in the presence of ligands which bind at the catalytic site. These studies support the existence of an ODCase structural domain which contains the ODCase catalytic site and a dimerization surface of UMP synthase, but the domain may not have the regulatory site required to form the altered dimer form.
Langdon
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SD
Department of Biochemistry and Nutrition, School of Medicine, University of North Carolina, Chapel Hill 27514.
Jones
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ME
D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010430:Pentosyltransferases / Q000235:genetics*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D009097:Multienzyme Complexes; C027157:uridine 5'-monophosphate synthase; D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D002262:Carboxy-Lyases; D009964:Orotidine-5'-Phosphate Decarboxylase