Study of the kinetic and physical properties of the orotidine-5'-monophosphate decarboxylase domain from mouse UMP synthase produced in Saccharomyces cerevisiae.

Abstract

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.

• Authors: S D Langdon, M E Jones
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 262 Issue 27 Pg. 13359-65 (Sep 25 1987) ISSN: 0021-9258 [Print] United States
• PMID: 3308878 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Multienzyme Complexes
• uridine 5'-monophosphate synthase
• Pentosyltransferases
• Orotate Phosphoribosyltransferase
• Carboxy-Lyases
• Orotidine-5'-Phosphate Decarboxylase

Topics

• Base Sequence
• Carboxy-Lyases (genetics)
• Cloning, Molecular
• Escherichia coli (genetics)
• Kinetics
• Multienzyme Complexes (genetics, isolation & purification, metabolism)
• Orotate Phosphoribosyltransferase (genetics, isolation & purification, metabolism)
• Orotidine-5'-Phosphate Decarboxylase (genetics, isolation & purification, metabolism)
• Pentosyltransferases (genetics)
• Plasmids
• Saccharomyces cerevisiae (enzymology, genetics)