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The arginine-independent, de novo biosynthetic pathway of pyrimidines in Dictyostelium discoideum is initiated by a class II carbamoyl-phosphate synthetase (EC 6.3.5.5) specific for pyrimidine biosynthesis which utilized L-glutamine as its N donor and was partially inhibited by both UTP and CTP. The second step in the de novo pathway was provided by an unregulated aspartate transcarbamoylase (EC 2.1.3.2) which primarily appeared as a multimeric enzyme of 105 kilodaltons. The next enzyme, dihydroorotase (EC 3.5.2.3), was approximately 90-100 kilodaltons. Although the early enzymatic activities of the pyrimidine pathway appeared to reside in independent protein complexes, various unstable molecular species were observed. These structural variants may represent proteolytic fragments of a multienzyme complex. In addition to de novo synthesis, the amoeba demonstrated the capacity for salvage utilization of uracil, uridine, and cytidine. Upon starvation on a solid substratum, axenically grown amoebas began a concerted developmental program accompanied by a restructuring of nucleotide metabolism. The absolute levels of the ribonucleotide pools droppedby 98% within 30 h; however, both the adenylate energy charge and the GTP/ATP ratios were maintained for 50 h after the initiation of development. The maintenance of these metabolic energy parameters required the tight cell-cell contact necessary for development, and the capacity for pyrimidine metabolism was maintained throughout developmental morphogenesis.

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 Kms for PRPP and orotate were stoichiometric: 2.3 X 10(-6) M and 2.6 X 10(-6) M, respectively. Following separation, the Kms were significantly different: 1.3 X 10(-6) M for PRPP and 4.1 X 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 non-sequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.

1. The two polypeptide chains that comprise aspartate carbamoyltransferase in Escherichia coli are encoded by adjacent cistrons expressed in the order, promoter-leader-catalytic cistron-regulatory cistron (p-leader-pyrBI). These two cistrons and their single control region have been cloned as a 2,800 base pair (bp) fragment (The minimal coding requirement for the catalytic and regulatory polypeptides is about 1,350 bp plus control regions). The genes contained by this fragment are subject to normal repression controls and thus possess the intact control regions. 2. By deleting an internal fragment with specific restriction endonucleases, it was possible to construct shortened fragments which no longer produced the regulatory polypeptide. In these cases the expression of the catalytic cistron was normal and subject to repression upon growth in the presence of uracil. Since the pyrB cistron retained transcriptional control, the regulatory polypeptide was not required for expression or control of the catalytic cistron. As expected, the catalytic trimer (Mr = 100,000 daltons) from these deletion mutants had no effector response nor did it exhibit homotropic kinetics for aspartate. The enzyme was identical to the c3 trimer purified from the native holoenzyme by neohydrin dissociation. 3. Insertion of Mu d1(lac Apr) into the structural region of pyrB had a negative effect on the expression of pyrI. This supports the idea that the catalytic and regulatory polypeptide chains of aspartate carbamoyl-transferase are encoded by a single bicistronic operon. Detailed restriction analysis of the cloned pyrBI region has produced a genetic map of restriction sites which is colinear with the published amino acid sequences of the two polypeptides. These maps indicate that the 3'-terminus of the catalytic cistron is adjacent to the 5'-terminus of the regulatory cistron and separated by 10-20 bp. 4. DNA sequence analysis of the 5'-proximal regions of pyrBI revealed that an extensive leader sequence separated the promoter and first structural gene pyrB. This leader of approximately 150 bp contains an attenuator sequence and the translational signals required for the production of a leader polypeptide of 43 amino acids. In this paper we describe the structural organization of pyrBI, and provide a detailed analysis of its regulatory region including its DNA sequence.

The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction. The method provides for simple, accurate, and sensitive measurement of enzyme activity. The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used. Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately. The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.

The coordinate expression of four sequential enzymes in the de novo pyrimidine pathway may result from the interaction of the various polypeptides of the pathway in Serratia marcescens rather than represent some unit of transcriptional regulation. These interactions were defined by examining the polypeptide association observed in extracts of parental and mutant strains in a series of pleiotropic pyrimidine auxotrophs. Extracts of pyrE auxotrophs [processing dihydroorotate (DHOase) activity but no orotidine-5'-monophosphate pyrophosphorylase (OMPppase) activity] stimulate OMPppase activity in extracts of pyrC auxotrophs (posessing reduced OMPppase activity but no DHOase activity). Separation by molecular weight on Sephadex G200 has suggested an aggregation between the final two enzymes, OMPppase and OMPdecarboxylase (OMPdecase), and the earlier enzyme, DHOase. The reduction of OMPppase activity in pyrC auxotrophs (encoding either a defective polypeptide or reduced levels) is explained by the lack of adequate levels of DHOase for aggregate formation. Such polypeptide interactions appear to mimic the coordinate formation of polypeptides which are controlled as a unit of regulation. The measurable levels of enzymatic activity vary in a quantitatively identical manner, but the variation does not result directly from the regulation of polypeptide formation.

The reaction mechanism for the phosphotriesterase from Pseudomonas diminuta has been examined. When paraoxon (diethyl 4-nitrophenyl phosphate) is hydrolyzed by this enzyme in oxygen-18-labeled water, the oxygen-18 label is found exclusively in the diethyl phosphate product. The absolute configurations for the (+) and (-) enantiomers of O-ethyl phenylphosphonothioic acid have been determined by X-ray diffraction structural determination of the individual crystalline 1-phenylethylamine salts. The (+) enantiomer of the free acid corresponds to the RP configuration. The RP enantiomer of O-ethyl phenylphosphonothioic acid has been converted to the SP enantiomer of EPN [O-ethyl O-(4-nitrophenyl) phenylphosphonothioate]. (SP)-EPN is hydrolyzed by the phosphotriesterase to the SP enantiomer of O-ethyl phenylphosphonothioic acid. The enzymatic reaction therefore proceeds with inversion of configuration. These results have been interpreted as an indication of a single in-line displacement by an activated water molecule directly at the phosphorus center of the phosphotriester substrate. (RP)-EPN is not hydrolyzed by the enzyme at an appreciable rate.

The effect of simulated saturated diving environments on mutagenicity was investigated using two histidine auxotrophs (hisG46 and hisTA1950 derived by Ames from Salmonella typhimurium LT2) exposed to several chemical mutagens. In agar diffusion tests with impregnated paper discs, 68 atm of normoxic supporting gases (containing 0.2 atm O2) reduced the number of histidine revertants of auxotrophs exposed to N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and two other alkylating agents. The effect was induced in increasing order by helium, a mixture of neon and helium, nitrogen and argon. The effect of the gases on the growth of the two strains was small and did not explain the reduction of revertant recovery. On the other hand, it was shown that NTG in concentrations of 0.6 to 250 mug/ml was significantly more lethal for the hisG46 strain under 68 and 136 atm than at 1 atm. Revertant recovery was similarly related in both conditions. The maximal number of revertants, approx. 2.6 X 10(4), was obtained at 136 atm when the concentration of NTG was 0.6--1.25 mug/ml and at 1 atm when the concentration of NTG was 5--10 mug/ml. A comparable hydrostatic environment did not affect revertant recovery. The most plausible interpretation of these results is that hyperbaric environments accelerate the rate of intracellular accumulation of NTG, which is manifest in greater mutagenicity at the lower concentrations and greater lethality at all concentrations.

The polar domains of the two transcarbamoylases, aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase, (OTCase) from Escherichia coli bind the common substrate carbamoyl phosphate and share extensive amino-acid sequence homology. The equatorial domains of the two enzymes differ in their substrate specificity (ATCase binds aspartate, OTCase binds ornithine) and have decreased sequence identity. While addressing the conservation of specific protein interactions during the evolution of these enzymes, we were able to switch one of their amino-acid-specific equatorial domains to produce a viable chimaeric enzyme. This was achieved by the in vitro fusion of DNA encoding the polar domain of OTCase to DNA encoding the equatorial domain of ATCase. The resulting gene fusion successfully transformed an argI-pyrB deletion strain of E. coli to pyrimidine prototrophy, giving rise to Pyr+ transformants that expressed ATCase but not OTCase activity. The formation of this active chimaeric enzyme shows that by exchanging protein domains between two functionally divergent enzymes we have achieved a switching in substrate specificity.