Pyrimidine biosynthesis in Pseudomonas veronii and its regulation by pyrimidines

Pyrimidine biosynthesis in the nutritionally versatile bacterium Pseudomonas veronii ATCC 700474 appeared to be controlled by pyrimidines. When wild type cells were grown on glucose in the presence of uracil, four enzyme activities were depressed while all five enzyme activities increased in succinate-grown cells supplemented with uracil. Independent of carbon source, orotic acid-grown cells elevated aspartate transcarbamoylase, dihydroorotase, orotate phosphoribosyltransferase or OMP decarboxylase activity. Pyrimidine limitation of glucose-grown pyrimidine auxotrophic cells lacking OMP decarboxylase activity resulted in at least a doubling of the enzyme activities relative to their activities in uracil-grown cells. Less derepression of the enzyme activities was observed after pyrimidine limitation of succinate-grown mutant cells possibly due to catabolite repression. Aspartate transcarbamoylase activity in Ps. veronii was regulated at the level of enzyme activity since the enzyme was strongly inhibited by pyrophosphate, UDP, UTP, ADP, ATP and GTP. Overall, the regulation of pyrimidine biosynthesis in Ps. veronii could be used to differentiate it from other taxonomically related species of Pseudomonas.     

Regulation of arginine and pyrimidine biosynthesis in Pseudomonas putida.

Repression of biosynthetic enzyme synthesis in Pseudomonas putida is incomplete even when the bacteria are growing in a nutritionally complex environment. The synthesis of four of the enzymes of the arginine biosynthetic pathway (N-acetyl-alpha-glutamokinase/N-acetylglutamate-gamma-semialdehyde dehydrogenase, ornithine carbamoyltransferase and acetylornithine-delta-transaminase) could be repressed and derepressed, but the maximum difference observed between repressed and derepressed levels for any enzyme of the pathway was only 5-fold (for ornithine carbamoyltransferase). No repression of five enzymes of the pyrimidine biosynthetic pathway (aspartate carbamoyltransferase, dihydro-orotase, dihydro-orotate dehydrogenase, orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase) could be detected on addition of pyrimidines to minimal asparagine cultures of P. putida A90, but a 1-5- to 2-fold degree of derepression was found following pyrimidine starvation of pyrimidine auxotrophic mutants of P. putida A90. Aspartate carbamoyltransferase in crude extracts of P. putida A90 was inhibited in vitro by (in order of efficiency) pyrophosphate, CTP, UTP and ATP, at limiting but not at saturating concentrations of carbamoyl phosphate.     

Regulation of the Pyrimidine Biosynthetic Pathway in <Emphasis Type="Italic">Pseudomonas mucidolens</Emphasis>

Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.     

Regulation of Salmonella typhimurium pyr gene expression: effect of changing both purine and pyrimidine nucleotide pools

The synthesis of the pyrimidine biosynthetic enzymes is repressed by the pyrimidine nucleotide end-products of the pathway. However, purine nucleotides also play a role. In this study, I have measured expression of the pyr genes (pyrA-E) in Salmonella typhimurium strains harbouring mutations that permit manipulation of the intracellular pools of both pyrimidine and purine nucleotides. The results identify the effectory purine compound as being a guanine nucleotide; it is probably GTP, but it may be GDP or GMP. The synthesis of carbamoylphosphate synthase, encoded by pyrA, and particularly dihydroorotase, encoded by pyrC, and dihydroorotate dehydrogenase, encoded by pyrD, is stimulated by the guanine nucleotide, while the synthesis of aspartate transcarbamoylase, encoded by pyrBI, and orotate phosphoribosyltransferase, encoded by pyrE, is inhibited by guanine nucleotides. The regulatory pattern of each pyr gene is discussed in relation to present knowledge on gene structure and regulatory mechanism.     

Pyrimidine synthesis in Neurospora crassa: regulation of enzyme activities

The regulation of several enzymes involved in pyrimidine biosynthesis in Neurospora crassa has been studied. Elevation of ATCase (l-aspartate carbamoyltransferase) activity is found in all pyrimidine-requiring mutants when they are starved for uridine. DHOase (dihydroorotase) is an unstable enzyme, and it is impossible to conclude what type of regulation, if any, controls this enzyme. DHOdehase (dihydroorotate dehydrogenase) activity shows a marked elevation in uridine-starved pyr-2 cultures, a mutant blocked late in the pathway. Several mutants blocked early in the pathway show much smaller increases in DHOdehase activity and possible explanations for this are discussed. Differences in the modes of regulation of the pyrimidine biosynthetic pathways in various organisms are compared.     

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha ATCC 9890 was investigated and the pyrimidine biosynthetic pathway enzyme activities were affected by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. In pyrimidine-grown ATCC 9890 cells, the activities of four de novo enzymes could be depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the succinate-grown mutant strain cells, pyrimidine limitation of this strain caused dihydroorotase activity to increase about 3-fold while dihydroorotate dehydrogenase and orotidine 5'-monophosphate decarboxylase activities rose about 2-fold. Regulation of de novo pathway enzyme synthesis by pyrimidines appeared to be occurring. At the level of enzyme activity, aspartate transcarbamoylase activity in P. synxantha ATCC 9890 was strongly inhibited in vitro by pyrophosphate, UTP, ADP, ATP, CTP and GTP under saturating substrate concentrations.     

Pyrimidine biosynthetic enzymes of Salmonella typhimurium, repressed specifically by growth in the presence of cytidine.

The repressive effects of exogenous cytidine on growing cells was examined in a specially constructed strain in which the pool sizes of endogenous uridine 5'-diphosphate and uridine 5'-triphosphate cannot be varied by the addition of uracil and/or uridine to the medium. Five enzymes of the pyrimidine biosynthetic pathway and one enzyme of the arginine biosynthetic pathway were assayed from cells grown under a variety of conditions. Cytidine repressed the synthesis of dihydroorotase (encoded by pyrC), dihydroorotate dehydrogenase (encoded by pyrD), and ornithine transcarbamylase (encoded by argI). Moreover, aspartate transcarbamylase (encoded by pyrB) became further derepressed upon cytidine addition, whereas no change occurred in the levels of the last two enzymes (encoded by pyrE and pyrF) of the pyrimidine pathway. Quantitative nucleotide pool determinations have provided evidence that any individual ribo- or deoxyribonucleoside mono-, di-, or triphosphate of cytosine or uracil is not a repressing metabolite for the pyrimidine biosynthetic enzymes. Other nucleotide derivatives or ratios must be considered.     

Cloning and expression of malarial pyrimidine enzymes

We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from Plasmodium falciparum and sequence information from the Malarial Genome Project. Genes encoding dihydroorotase (reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) have been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C-terminus and the enzymes were expressed in Escherichia coli. To overcome the toxicity of malarial OMP decarboxylase when expressed in E. coli, and the unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low levels of T7 lysozyme to inhibit T7 RNA polymerase used for recombinant expression, and extra copies of rare tRNAs. Catalytically-active OMP decarboxylase has been purified in tens of milligrams by chromatography on Ni-NTA. The gene encoding orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N-terminus when compared with sequences for this enzyme from other organisms. We have found that other pyrimidine enzymes also contain unusual protein inserts. Milligram quantities of pure recombinant malarial enzymes from the pyrimidine pathway will provide targets for development of novel antimalarial drugs.     

Organization of the pathway of de novo pyrimidine nucleotide biosynthesis in pea (Pisum sativum L. cv Progress No. 9) leaves

The organization of the enzymes of de novo pyrimidine nucleotide biosynthesis in pea (Pisum sativum L. cv Progress No. 9) has been studied. The first three enzymes of the pathway, carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, and dihydroorotase, are readily separable from one another; they are not part of a multifunctional complex. The final two activities of the pathway, orotate phosphoribosyltransferase and orotidylate decarboxylase, copurify and appear to be complexed in vivo. This organizational pattern is distinct from those reported for bacteria, yeast, and mammals. The differences in organization, in a pathway which is present in all organisms, make the pyrimidine biosynthetic pathway a very interesting candidate for evolutionary studies.     

Evolutionary implications of the mosaic pyrimidine-biosynthetic pathway in eukaryotes

The de-novo pyrimidine biosynthetic pathway involves six enzymes, in order from the first to the sixth step, carbamoyl-phosphate synthetase II (CPS II) comprising glutamine amidotransferase (GAT) and carbamoyl-phosphate synthetase (CPS) domains or subunits, aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase (DHOD), orotate phosphoribosyltransferase (OPRT), and orotidine-5'-monophosphate decarboxylase (OMPDC). In contrast with reports on molecular evolution of the individual enzymes, we attempted to draw an evolutionary picture of the whole pathway using the protein phylogeny. We demonstrate highly mosaic organizations of the pyrimidine biosynthetic pathway in eukaryotes. During evolution of the eukaryotic pathway, plants and fungi (or their ancestors) in particular may have secondarily acquired the characteristic enzymes. This is consistent with the fact that the organization of plant enzymes is highly chimeric: (1) two subunits of CPS II, GAT and CPS, cluster with a clade including cyanobacteria and red algal chloroplasts, (2) ACT not with a cyanobacterium, Synechocystis spp., irrespective of its putative signal sequence targeting into chloroplasts, and (3) DHO with a clade of proteobacteria. In fungi, DHO and OPRT cluster respectively with the corresponding proteobacterial counterparts. The phylogenetic analyses of DHOD and OMPDC also support the implications of the mosaic pyrimidine biosynthetic pathway in eukaryotes. The potential importance of the horizontal gene transfer(s) and endosymbiosis in establishing the mosaic pathway is discussed.     

Control of Pyrimidine Biosynthesis in Pseudomonas aeruginosa

The pathway of pyrimidine biosynthesis in Pseudomonas aeruginosa has been shown to be the same as in other bacteria. Twenty-seven mutants requiring uracil for growth were isolated and the mutant lesions were identified. Mutants lacking either dihydroorotic acid dehydrogenase, orotidine monophosphate pyrophosphorylase, orotidine monophosphate decarboxylase, or aspartic transcarbamylase were isolated; none lacking dihydroorotase were found. By using transduction and conjugation, four genes affecting pyrimidine biosynthetic enzymes have been identified and shown to be unlinked to each other. The linkage of pyrB to met-28 and ilv-2 was shown by contransduction. Repression by uracil alone or by broth could not be demonstrated for any enzymes of this pathway, in contrast to the situation in Escherichia coli and Serratia marcescens. In addition, derepression of these enzymes could not be demonstrated. A low level of feedback inhibition of aspartic transcarbamylase was found to occur. It is suggested that the control of such constitutive biosynthetic enzymes in P. aeruginosa may be related to the comprehensive metabolic activities of this organism.     

Isolation of uracil auxotrophs from Burkholderia cepacia ATCC 25416

K. LI AND T.P. WEST. 1995. Two uracil auxotrophs of the phytopathogen Burkholderia cepacia ATCC 25416, which is known to be involved in food spoilage, were isolated by a combination of ethylmethane sulphonate and D-cycloserine counterselection. One mutant exhibited depressed orotate phosphoribosyltransferase activity while the other mutant lacked orotidine 5'-monophosphate decarboxylase activity. Pyrimidine limitation of either auxotroph elevated aspartate transcarbamoylase and dihydroorotase activities by at least 1.5-fold indicating that these pathway enzymes may be repressible by a uracil-related compound in B. cepacia . Overall, regulation of de novo pyrimidine synthesis in the uracil auxotrophs of B. cepacia ATCC 25416 was observed.     

Subcellular localization of the pathway of de novo pyrimidine nucleotide biosynthesis in pea leaves

The subcellular distribution of the enzymes of de novo pyrimidine nucleotide biosynthesis was investigated in pea (Pisum sativum L. cv Progress No. 9) leaves. Aspartate carbamoyltransferase, the committed step of the pathway, was found to be strictly confined to the chloroplasts. Dihydro-orotase, orotate phosphoribosyl transferase, and orotidine decarboxylase activities were also found only in the plastids. The remaining enzyme of the pathway, dihydroorotate dehydrogenase, was shown to be mitochondrial.     

Cloning and Expression of Malarial Pyrimidine Enzymes

We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from Plasmodium falciparum and sequence information from the Malarial Genome Project. Genes encoding dihydroorotase (reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) have been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C‐terminus and the enzymes were expressed in Escherichia coli. To overcome the toxicity of malarial OMP decarboxylase when expressed in E. coli, and the unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low levels of T7 lysozyme to inhibit T7 RNA polymerase used for recombinant expression, and extra copies of rare tRNAs. Catalytically‐active OMP decarboxylase has been purified in tens of milligrams by chromatography on Ni‐NTA. The gene encoding orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N‐terminus when compared with sequences for this enzyme from other organisms. We have found that other pyrimidine enzymes also contain unusual protein inserts. Milligram quantities of pure recombinant malarial enzymes from the pyrimidine pathway will provide targets for development of novel antimalarial drugs.     

Control of the pyrimidine biosynthetic pathway in Pseudomonas pseudoalcaligenes

The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440. A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine. Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement. When the wildtype strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed. Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil. It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines. In vitro regulation of aspartate transcarbamoylase activity in P. pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by P_i, PP_i, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP.     

Glutamine-dependent carbamoyl-phosphate synthetase and control of pyrimidine biosynthesis in the parasitic helminth Schistosoma mansoni.

1. Carbomoyl-phosphate synthetase in Schistosoma mansoni utilizes L-glutamine as well as ammonia as nitrogen donor but does not require N-acetyl-L-glutamate for the activity. 2. The enzyme activity was inhibited by UDP, UTP, ADP and AMP, among which UDP was the most effective. 3. Aspartate carbamoyltransferase and dihydroorotase were also found and copurified with the synthetase. 4. Relative activities among these three enzymes were 1:30-60:3-8 throughout the purification. 5. These results suggest that the synthetase plays a key role in the control of pyrimidine biosynthesis de novo.     

Site of Synthesis of the Enzymes of the Pyrimidine Biosynthetic Pathway in Oat (Avena sativa L.) Leaves

Heat-bleached oat (Avena sativa L. cv Porter) leaves lacking 70S chloroplast ribosomes have been used to demonstrate that four chloroplast-localized enzymes of pyrimidine nucleotide biosynthesis: aspartate carbamoyl-transferase, dihydroorotase, orotidine phosphoribosyl-transferase, and orotidine-5′-phosphate decarboxylase, are synthesized on cytoplasmic ribosomes. Two other chloroplast enzymes, carbamoyl phosphate synthetase, involved in both pyrimidine and arginine biosynthesis, and ornithine carbamoyltransferase, an enzyme of arginine biosynthesis, were also shown to be made on 80S ribosomes.