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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.