Abstract
Orotidine-5′-monophosphate pyrophosphorylase (OMPppase, E.C. 2.4.2.10) and orotidylate decarboxylase (OMPdecase, E.C. 4.1.1.23) were purified from Serratia marcescens HY. These enzymes required physical association for maximal catalytic activities and formed a fragile complex with dihydroorotase (DHOase, E.C. 3.5.2.3.). OMPppase reversibly lost 50% of its activity upon separation from DHOase. The kinetic characteristics of OMPppase were modified by this separation. In the presence of DHOase, the K ms for PRPP and orotate were stoichiometric: 2.3×10−6 m and 2.6×10−6 m, respectively. Following separation, the K ms were significantly different: 1.3 × 10−6 m for PRPP and 4.1×10−6 m for orotate. OMPppase and OMPdecase could be reversibly separated by acrylamide gel electrophoresis, but the separation was accompanied by a loss of catalytic efficiency for both enzymes. DHOase readily associated into multiple molecular forms and could not be purified. The DHOase-OMPppase-OMPdecase interactions demonstrate that a weakly aggregated, multifunctional enzyme complex participates in the biosynthesis of pyrimidine nucleotides in S. marcescens. This unique association of nonsequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aitken, D. M., Lue, P. F., and Kaplan, J. G. (1975). Kinetics and reaction mechanism of the carbamylphosphate synthetase of a multienzyme aggregate from yeast. Can. J. Biochem. 53721.
Appel, S. H. (1968). Purification and kinetic properties of brain orotidine 5′-phosphate decarboxylase. J. Biol. Chem. 2433924.
Beckwith, J. R., Pardee, A. B., Austrian, R., and Jacob, F. (1962). Coordination of the synthesis of the enzymes in the pyrimidine pathway. J. Mol. Biol. 5618.
Brown, G. K., Fox, R. M., and O'Sullivan, W. J. (1975). Interconversion of different molecular weight forms of human erythrocyte orotidylate decarboxylase. J. Biol. Chem. 2507352.
Calvo, J. M., and Fink, G. R. (1971). Regulation of biosynthetic pathways in bacteria and fungi. Ann. Rev. Biochem. 40943.
Caroline, D. F. (1969). Pyrimidine synthesis in Neurospora crassa: Gene-enzyme relationships. J. Bacteriol. 1001371.
Eakin, R. T., and Mitchell, H. K. (1969). A mitochondrial oxidase system in Neurospora crassa. Arch. Biochem. Biophys. 134160.
Fausto, N. (1969). The control of RNA synthesis during liver regeneration: OMP pyrophosphorylase and decarboxylase activities in normal and regenerating liver. Biochim. Biophys. Acta 18266.
Fox, I. H., and Kelley, W. N. (1971). Human phosphoribosyl-pyrophosphate synthetase. J. Biol. Chem. 2465739.
Freiden, C. (1971). Protein-protein interaction and enzymatic activity. Ann. Rev. Biochem. 40653.
Gerhart, J. C., and Pardee, A. B. (1962). The enzymology of control by feedback inhibition. J. Biol. Chem. 237891.
Ginsburg, A., and Stadtman, E. R. (1970). Multienzyme systems. Ann. Rev. Biochem. 39429.
Grobner, W., and Kelley, W. N. (1975). Effect of allopurinol and its metabolic derivatives on the configuration of human orotate phosphoriboxyltransferase and orotidine 5′-phosphate decarboxylase. Biochem. Pharmacol. 24379.
Hutson, J. Y., and Downing, M. (1968). Pyrimidine biosynthesis in Lactobacillus leichmannii. J. Bacteriol. 961249.
Isaac, J. H., and Holloway, B. W. (1968). Control of pyrimidine biosynthesis in Pseudomonas aeruginosa. J. Bacteriol. 961732.
Jones, M. E. (1971). Regulation of pyrimidine and arginine biosynthesis in mammals. Adv. Enzyme Regul. 919.
Jones, M. E. (1972). Regulation of uridylic acid biosynthesis in eukaryotic cells. Cur. Top. Cell. Regul. 6227.
Karibian, D., and Couchoud, P. (1974). Dihydro-orotate oxidase of Escherichia coli K12: Purification, properties, and relation to the cytoplasmic membrane. Biochim. Biophys. Acta 364218.
Kasbekar, E. G., Nagabhushanam, A., and Greenberg, D. M. (1964). Purification and properties of orotic acid-decarboxylating enzymes from calf thymus. J. Biol. Chem. 2394245.
Labrum, E. L., and Bunting, M. I. (1953). Spontaneous and induced color variation of the HY strain of Serratia marcescens. J. Bacteriol. 65394.
LaCroute, F. (1968). Regulation of pyrimidine biosynthesis in Saccharomyces cerevisiae. J. Bacteriol. 95825.
Lieberman, I., Kornberg, A., and Simms, E. S. (1954). Enzymatic synthesis of pyrimidine and purine nucleotides. II. Orotidine-5′-phosphate pyrophosphorylase and decarboxylase. J. Amer. Chem. 762844.
Lieberman, I., Kornberg, A., and Simms, E. S. (1955). Enzymatic synthesis of pyrimidine nucleotides orotidine-5′-phosphate and uridine-5′-phosphate. J. Biol. Chem. 215403.
Long, C. W., Levitzki, A., and Koshland, D. E., Jr. (1970). The subunit interactions of cytidine triphosphate synthetase. J. Biol. Chem. 24580.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193265.
Matsuura, T., and Jones, M. E. (1974). Subcellular localization in the Ehrlich ascites cell of the enzyme which oxidizes dehydroorotate to orotate. In Richter, D. (ed.), The Lipmann Symposium: Energy, Regulation and Biosynthesis in Molecular Biology, Walter de Gruyter, Berlin, pp. 422–434.
Miller, R. W. (1975). A high molecular weight dihydroorotate dehydrogenase of Neurospora crassa. Purification and properties of this enzyme. Can. J. Biochem. 531288.
Miller, R. W., and Curry, J. R. (1969). Mammalian dihydroorotate-ubiquinone reductase complex. II. Correlation with cytochrome oxidase, mode of linkage with the cytochrome chain, and general properties. Can. J. Biochem. 47725.
Mori, M., and Tatibana, M. (1973). Dissociation by elastase digestion of enzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver. Biochem. Biophys. Res. Commun. 541525.
Mori, M., Ishida, H., and Tatibana, M. (1975). Aggregation states and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver. Biochemistry 142622.
Nagano, Y., Samjima, H., and Kinoshita, S. (1966). Conversion of orotic acid to uridine-5′-monophosphate by enzymes of Micrococcus glutamicus. Agr. Biol. Chem. 3083.
O'Donovan, G. A., and Neuhard, J. (1970). Pyrimidine metabolism in microorganisms. Bacteriol. Rev. 34278.
Ornstein, L. (1961). Disc Electrophoresis, Canal Industrial Corp. Bethesda, Md.
Potvin, B. W., Kelleher, R. J., Jr., and Gooder, H. (1975). Pyrimidine biosynthetic pathway of Bacillus subtilis. J. Bacteriol. 123604.
Pynadath, T. L., and Fink, R. M. (1967). Studies of orotidine-5′-phosphate decarboxylase in Neurospora crassa. Arch. Biochem. Biophys. 118185.
Reichard, P. (1959). The enzymic synthesis of pyrimidines. Adv. Enzymol. 21263.
Shoaf, W. T., and Jones, M. E. (1973). Uridylic acid synthesis in Ehrlich ascites carcinoma: Properties, subcellular distribution, and nature of enzyme complexes of the six biosynthetic enzymes. Biochemistry 124039.
Umezu, K., Amaya, T., Yoshimoto, A., and Tomita, K. (1971). Purification and properties of orotidine-5′-phosphate pyrophosphorylase and orotidine-5′-phosphate decarboxylase from baker's yeast. J. Biochem. 70249.
Warburg, O., and Christian, W. (1942). Distributed by Calbiochem. Biochem. Z. 310384.
Wild, J. R., and Belser, W. L. (1977). Pyrimidine biosynthesis in Serratia marcescens: A possible role for nonsequential enzyme interactions in mimicking coordinate gene expression. Biochem. Genet. 15157.
Author information
Authors and Affiliations
Additional information
This work was supported by grants from the National Science Foundation (NSF GB 5811) and the Office of Naval Research (Nonr 4413). One of us (J.W.) was a National Science Foundation Graduate Fellow.
Rights and permissions
About this article
Cite this article
Wild, J.R., Belser, W.L. Pyrimidine biosynthesis in Serratia marcescens: Polypeptide interactions of three nonsequential enzymes. Biochem Genet 15, 173–193 (1977). https://doi.org/10.1007/BF00484560
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00484560