Search

Taylor , W. H. (Portland State College, Portland, Ore.), M. L. Taylor, and D. F. Eames . Two functionally different dihydroorotic dehydrogenases in bacteria. J. Bacteriol. 91: 2251–2256. 1966.—We have investigated the relationship between the two kinds of dihydroorotic dehydrogenases produced by bacteria. A pseudomonad, capable of growth on a salts medium with glucose, aspartate, glycerol, or orotate as the carbon source, was isolated from lake bank mud. A particle-bound dihydroorotic dehydrogenase, similar to the biosynthetic enzyme in Escherichia coli , was formed by the pseudomonad when the carbon source was orotate, glucose, glycerol, or aspartate. A soluble, degradative nicotinamide adenine dinucleotide phosphate-linked dihydroorotic dehydrogenase, as well as the particle-bound biosynthetic enzyme, was formed when the pseudomonad was cultivated on orotate. The biosynthetic enzyme links to oxygen or ferricyanide, but not to pyridine nucleotides. Zymobacterium oroticum , when cultivated on glucose, contained only the biosynthetic type of dihydroorotic dehydrogenase. The presence of two functionally different dihydroorotic dehydrogenases in the pseudomonad was suggested on the basis of the following observations: (i) the two enzyme activities were separated by centrifugation; (ii) the pyridine nucleotide-linked activity was formed only when orotate was present in the growth medium; and (iii) the biosynthetic enzyme was stable to storage at −20 C for 4 months, whereas the degradative enzyme activity was destroyed by storage under these conditions.

Taylor, W. Herman (Portland State College, Portland, Ore.), and Mary L. Taylor . Enzymes of the pyrimidine pathway in Escherichia coli . II. Intracellular localization and properties of dihydroorotic dehydrogenase. J. Bacteriol. 88: 105–111. 1964.—Intracellular localization of three enzymes of the pyrimidine pathway in Escherichia coli was studied. Dihydroorotic dehydrogenase was found to be associated with the membrane portion of lysed spheroplasts. Centrifugal fractionation of cell-free extracts showed all the dihydroorotic dehydrogenase activity to be associated with large structures, probably cell wall-membrane fragments. In contrast, all orotidylic decarboxylase activity was found in the cytoplasm in both lysed spheroplasts and cell-free extracts. Aspartate transcarbamylase activity appeared to be particulate in repressed cells, but only 25% was particulate in derepressed cells. Dihydroorotic dehydrogenase was shown to be bound to oxidative particles by oxygen uptake and orotate production from dihydroorotate. A ferricyanide reduction assay, suitable for measuring soluble and particulate enzyme, was devised for dihydroorotic dehydrogenase. Soluble dihydroorotic dehydrogenase was prepared by use of deoxycholate. A 20-fold purification of the enzyme compared to whole-cell activity was achieved by ammonium sulfate fractionation of the deoxycholate-soluble enzyme. Although cytochromes were implicated by cyanide inhibition of aerobic orotate production by particles, the purified enzyme appeared to be separated from the cytochromes, as shown by lack of cyanide inhibition in the ferricyanide assay. The purified soluble enzyme did not react in the aerobic assay previously used by others for assay of this enzyme. In contrast to the degradative dihydroorotic dehydrogenases reported by other workers, the biosynthetic dihydroorotic dehydrogenase of E. coli did not link to pyridine nucleotides.

Taylor, W. Herman (Portland State College, Portland, Ore.), and G. David Novelli . Enzymes of the pyrimidine pathway in Escherichia coli . I. Synthesis by cells and spheroplasts. J. Bacteriol. 88: 99–104. 1964.—Upon release from repression, cells and spheroplasts of two mutants of Escherichia coli efficiently synthesized aspartate transcarbamylase and ornithine transcarbamylase, whereas only cells synthesized dihydroorotic dehydrogenase. Ethylenediaminetetraacetate treatment and sucrose incubation of cells were found to be responsible for the loss of dihydroorotic dehydrogenase synthesis. Spheroplasts required the addition of amino acids and an energy source for the synthesis of aspartate transcarbamylase. Uracil repressed synthesis of aspartate transcarbamylase in spheroplasts as well as in cells. Chloramphenicol inhibition and amino acid requirement for increased aspartate transcarbamylase activity in spheroplasts indicated de novo protein synthesis. E. coli 15, R185-482, and E. coli K-12, 496, were used to study the effect of carbon source and stimulation by orotate and dihydroorotate on synthesis of dihydroorotic dehydrogenase. Only E. coli 15, R185-482, showed any stimulation of dihydroorotic dehydrogenase synthesis. When glucose was the carbon source, orotate but not dihydroorotate stimulated; with glycerol as carbon source, dihydroorotate stimulated and orotate acted as a repressor. These results are discussed in terms of induction and pyrimidine supply to the cells.

Some of the catalytic properties of the biosynthetic dihydroorotate dehydrogenase purified from an anaerobic bacterium, Lactobacillus bulgaricus , are described. Studies with p -hydroxymercuribenzoate, N -ethylmaleimide, and mercuric chloride showed that sulfhydryl groups are necessary for transfer of electrons from dihydroorotate to a variety of electron acceptors. Protection studies with substrates for the enzyme indicated that free sulfhydryl groups at or near the active center are required for catalytic activity. Evidence is presented for the production of superoxide free radicals during reaction of the enzyme with molecular oxygen. Inhibitor studies with Tiron indicated that reduction of cytochrome c by the enzyme may involve the superoxide free radical as an intermediate. Orotate, one of the substrates for the enzyme, has been found to be a competitive inhibitor for the dihydroorotate site. The K i for orotate as estimated by several techniques is 0.1 mM. The K m for dihydroorotate with ferricyanide as the electron acceptor is estimated to be 0.5 mM.

This paper describes the first detailed study on a dihydroorotate dehydrogenase involved in pyrimidine biosynthesis. In most organisms the enzyme is membrane-bound; however, a soluble dihydroorotate dehydrogenase was produced in relatively high levels when the anaerobe, Lactobacillus bulgaricus , was released from repression. The enzyme was purified 213-fold over derepressed levels with a 39% recovery of enzyme units. The enzyme showed only one minor protein contaminant when analyzed by polyacrylamide electrophoresis. It was characterized as a flavoprotein containing only flavine mononucleotide as the prosthetic group. Molecular weight estimations by gel filtration gave a value of approximately 55,000, which is one-half that of the degradative enzyme described by others. During aerobic oxidation of dihydroorotate, the rates of oxygen consumption, orotate formation, and hydrogen peroxide formation were equal, as would be expected in a flavoprotein-catalyzed reaction. The enzymatic activity with ferricyanide as acceptor was optimum around p H 7.7. The stimulation of enzymatic activity over a wide p H range by ammonium sulfate was attributed to an effect on the maximum velocity of the reaction. As analyzed by polyacrylamide electrophoresis, inactivation of the enzyme by visible light resulted in the appearance of a second protein band with lowered specific activity. The purified enzyme used redox dyes, oxygen, or cytochrome c as electron acceptors but was not active with pyridine nucleotides. Flavine adenine dinucleotide has been implicated at the active site for pyridine nucleotide reduction in the degradative enzyme. The biosynthetic enzyme lacks this flavine and the associated activity.