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.     

Sequence of the URA1 gene encoding dihydroorotate dehydrogenase from the basidiomycete fungus Agrocybe aegerita.

The URA1 gene encoding dihydroorotate dehydrogenase (DHOdehase) from the edible basidiomycete, Agrocybe aegerita, has been cloned by complementation of the Escherichia coli pyrD mutation. The nucleotide sequence of a 1531-bp genomic fragment carrying URA1 revealed two uninterrupted open reading frames (ORFs) separated by 61 bp. The larger ORF can encode a 328-amino acid (aa) DHOdehase that has 53% homology with the corresponding protein from E. coli. Comparison with other DHOdehase aa sequences showed essentially conservation of the cofactor-binding site of flavoproteins.     

Generation of auxotrophic mutants of Enterococcus faecalis.

A 22-kb segment of chromosomal DNA from Enterococcus faecalis OG1RF containing the pyrimidine biosynthesis genes pyrC and pyrD was previously detected as complementing Escherichia coli pyrC and pyrD mutations. In the present study, it was found that the E. faecalis pyrimidine biosynthetic genes in this clone (designated pKV48) are part of a larger cluster resembling that seen in Bacillus spp. Transposon insertions were isolated at a number of sites throughout the cluster and resulted in loss of the ability to complement E. coli auxotrophs. The DNA sequences of the entire pyrD gene of E. faecalis and selected parts of the rest of the cluster were determined, and computer analyses found these to be similar to genes from Bacillus subtilis and Bacillus caldolyticus pyrimidine biosynthesis operons. Five of the transposon insertions were introduced back into the E. faecalis chromosome, and all except insertions in pyrD resulted in pyrimidine auxotrophy. The prototrophy of pyrD knockouts was observed for two different insertions and suggests that E. faecalis is similar to Lactococcus lactis, which has been shown to possess two pyrD genes. A similar analysis was performed with the purL gene from E. faecalis, contained in another cosmid clone, and purine auxotrophs were isolated. In addition, a pool of random transposon insertions in pKV48, isolated in E. coli, was introduced into the E. faecalis chromosome en masse, and an auxotroph was obtained. These results demonstrate a new methodology for constructing defined knockout mutations in E. faecalis.     

二氢乳清酸脱氢酶的结构特征和催化循环研究进展

二氢乳清酸脱氢酶是黄素依赖的线粒体酶,它催化嘧啶从头合成的第4步反应,将二氢乳清酸氧化为乳清酸。通过选择性抑制二氢乳清酸脱氢酶,从而抑制嘧啶的合成,已被开发用于治疗癌症、自身免疫性疾病、细菌或病毒感染以及寄生虫疾病等。抑制剂的开发需详细了解二氢乳清酸脱氢酶的结构特征和催化循环机制。因此,文中主要从这两个方面进行了综述,并展望了该酶的抑制剂在临床应用中的前景。     

Cloning of the URA1 gene of Saccharomyces cerevisiae

A 5.7-kilobase segment of Saccharomyces cerevisiae deoxyribonucleic acid which complements both the yeast ura1 and Escherichia coli pyrD mutations in dihydroorotate dehydrogenase has been cloned in plasmid YRp7.     

Toxicity of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate in Salmonella typhimurium

Growth of Salmonella typhimurium pyrC or pyrD auxotrophs was severely inhibited in media that caused derepressed pyr gene expression. No such inhibition was observed with derepressed pyrA and pyrB auxotrophs. Growth inhibition was not due to the depletion of essential pyrimidine biosynthetic pathway intermediates or substrates. This result and the pattern of inhibition indicated that the accumulation of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate was toxic. This intermediate is synthesized by the sequential action of the first two enzymes of the pathway encoded by pyrA and pyrB and is a substrate for the pyrC gene product. It should accumulate to high levels in pyrC or pyrD mutants when expression of the pyrA and pyrB genes is elevated. The introduction of either a pyrA or pyrB mutation into a pyrC strain eliminated the observed growth inhibition. Additionally, a direct correlation was shown between the severity of growth inhibition of a pyrC auxotroph and the levels of the enzymes that synthesize carbamyl aspartate. The mechanism of carbamyl aspartate toxicity was not identified, but many potential sites of growth inhibition were excluded. Carbamyl aspartate toxicity was shown to be useful as a phenotypic trait for classifying pyrimidine auxotrophs and may also be useful for positive selection of pyrA or pyrB mutants. Finally, we discuss ways of overcoming growth inhibition of pyrC and pyrD mutants under derepressing conditions.     

Identification of a novel gene of pyrimidine nucleotide biosynthesis, pyrDII, that is required for dihydroorotate dehydrogenase activity in Bacillus subtilis.

An in-frame deletion in the coding region of a gene of previously unidentified function (which is called orf2 and which we propose to rename pyrDII) in the Bacillus subtilis pyr operon led to pyrimidine bradytrophy, markedly reduced dihydroorotate dehydrogenase activity, and derepressed levels of other enzymes of pyrimidine biosynthesis. The deletion mutation was not corrected by a plasmid encoding pyrDI, the previously identified gene encoding dihydroorotate dehydrogenase, but was complemented by a plasmid encoding pyrDII. We propose that pyrDII encodes a protein subunit of dihydroorotate dehydrogenase that catalyzes electron transfer from the pyrDI-encoded subunit to components of the electron transport chain.     

Developmental control of the expression of the dihydroorotate dehydrogenase and UMP synthase genes in Dictyostelium discoideum

Developmental variations in the expression of two genes of the de novo pyrimidine biosynthetic pathway have been examined in Dictyostelium discoideum. One gene, DdPYR4, encodes the dihydroorotate dehydrogenase (EC 1.3.3.1); the other, DdPYR5-6, encodes the UMP synthase which in D. discoideum is a bifunctional enzyme harboring both the orotate phosphoribosyl transferase activity (EC 2.4.2.10) and the OMP decarboxylase activity (EC 4.1.1.23). The relative amount of mRNA for both genes has been estimated by hybridization with the previously cloned DNAs and compared with the amount of actin mRNA. The level of both mRNAs is dramatically reduced after 4 h of development and remains at a low level later in development. In contrast to these variations, the specific activity of the enzymes encoded by these genes during development is similar to that measured during exponential growth. These results lead us to propose that DdPYR4 and DdPYR5-6 genes encode for relatively stable proteins and that their synthesis is reduced to maintain a constant level of enzymes in non-growing cells. This mode of regulation could apply to a large number of housekeeping genes.     

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.     

Human spleen dihydroorotate dehydrogenase: a study of inhibition of the enzyme

Seventy-one pyrimidine analogs have been tested as possible inhibitors of human spleen mitochondrial dihydroorotate dehydrogenase. Of these nine were demonstrated to be effective inhibitors of the enzymic activity. Two compounds, dihydro-5-azaorotate and 6-thiobarbiturate appeared to be specific inhibitors of the DHO-DHase. In addition, three compounds, 5-azaorotate, 5-bromoorotate, and barbiturate were also inhibitory against the two subsequent enzymes of the pathway, orotate phosphoribosyltransferase and orotidylate decarboxylase, so that they could act against three enzymes of the mammalian pyrimidine de novo biosynthetic pathway.     

Cloning, expression, purification, and characterization of Leishmania major dihydroorotate dehydrogenase

Leishmania major Friedlin (LmjF) is a protozoan parasite whose genomic sequence has been recently elucidated. Here we have cloned, overexpressed, purified, and characterized the product of the gene from LmjF chromosome 16: LmjF16.0530, which encodes a protein with putative dihydroorotate dehydrogenase activity. Dihydroorotate dehydrogenase (DHODH) is a flavoprotein that catalyses the oxidation of L-dihydroorotate to orotate, the fourth sequential step in the de novo pyrimidine nucleotide synthesis pathway. The predicted enzyme from L. major was cloned and expressed in Escherichia coli strain BL21(DE3) as a histidine-tag fusion protein and purified to homogeneity using affinity chromatography. The final product was homogeneous in SDS-PAGE gel electrophoresis. The dihydroorotate oxidase activity has been assayed and the steady-state kinetic mechanism has been determined using fumarate as the oxidizing substrate. The catalysis by LmDHODH enzyme proceeds by a Ping-Pong Bi-Bi mechanism and the kinetic parameters Km were calculated to be 90 and 418 microM for dihydroorotate and fumarate, respectively, and Vmax was calculated to be 11 micromol min-1 mg-1. Our results confirmed that the product of the gene LmjF16.0530, whose function has previously been predicted based on homology to known proteins, can therefore be positively assigned as L. major DHODH.     

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.     

Dihydroorotate induces Ca2+ release from rat liver mitochondria: a contribution to the mechanism of alloxan-induced Ca2+ release

The present work deals with the effects of alloxan on rat liver mitochondria, involving formation of toxic oxygen derivatives and Ca2+ release, and its relations to a physiological pathway, pyrimidine biosynthesis, particularly dihydroorotate dehydrogenation. Ca2+ release by intact isolated mitochondria was studied and redox transfer from solubilized mitochondria to 2,6-dichloroindophenol in the presence of cyanide. In intact mitochondria 5mM dihydroorotate caused a Ca2+ efflux comparable to 2mM alloxan. Both effects were suppressed by orotate, a potent inhibitor of dihydroorotate dehydrogenase, and by ADP, an inhibitor of the alloxan effects. In lysed mitochondria orotate but not ADP inhibited ubiquinone-linked reduction of 2,6-dichloroindophenol with dihydroorotate and with alloxan in a concentration-dependent manner. It is concluded that in vitro part of the redox cycling of alloxan is catalysed by dihydroorotate dehydrogenase whereas the nonsuppressible part reacts nonenzymatically. Without ADP the respiratory control blocks the reoxidation of coenzyme Q via the respiratory chain, thus giving preference to the regeneration by artificial electron acceptors, e.g. oxygen, yielding superoxide radicals and hydrogen peroxide, a notorious inducer of Ca2+ release. In vivo the enzymatic reoxidation of reduced alloxan by dihydroorotate dehydrogenase may be superior to the non-enzymatic pathway since the nonenzymatic fraction of reoxidation decreases with decreasing alloxan concentration.     

PHYLOGENETIC IMPLICATIONS OF THE CAD COMPLEX FROM THE PRIMITIVE RED ALGA CYANIDIOSCHYZON MEROLAE (CYANIDIALES,RHODOPHYTA)1

The de novo pyrimidine biosynthetic pathway consists of six enzymes: carbamoyl‐phosphate synthetase II (CPS II), aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine‐5′‐monophosphate decarboxylase. The origin and organization of the first three enzymes differ markedly between Opisthokonta (Metazoa and Fungi) and the Amoebozoa and green plants. However, no information has been available regarding the characteristics of such genes in other photosynthetic eukaryotes. In this study, we examined the pyrimidine biosynthetic cluster in the primitive red alga Cyanidioschyzon merolae P. DeLuca et al. isolate 10D. Unlike the situation in green plants, the CPS II, ACT, and DHO of C. merolae were fused to form a single open reading frame (the CAD complex), as in the Opisthokonta and Amoebozoa. Phylogenetic analysis of the CPS domain sequences suggested that this red algal CAD complex did not result from a recent lateral gene transfer from Metazoa or Fungi but that the fusion of the three genes occurred before the divergence of Opisthokonta, Amoebozoa, and the red algae. These results cast doubt on the recent hypothesis that the Opisthokonta and Amoebozoa form a monophyletic group, based on the presence in both of the CAD complex.     

The Origin of Dihydroorotate Dehydrogenase Genes of Kinetoplastids, with Special Reference to Their Biological Significance and Adaptation to Anaerobic, Parasitic Conditions

Trypanosoma cruzi dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the de novo pyrimidine biosynthetic pathway, is localized in the cytosol and utilizes fumarate as electron acceptor (fumarate reductase activity), while the enzyme from other various eukaryotes is mitochondrial membrane-linked. Here we report that DHOD-knockout T. cruzi did not express the enzyme protein and could not survive even in the presence of pyrimidine nucleosides, substrates for the potentially active salvage pathway, suggesting a vital role of fumarate reductase activity in the regulation of cellular redox balance. Cloning and phylogenetic analysis of euglenozoan DHOD genes showed that the euglenoid Euglena gracilis had a mitochondrial DHOD and that biflagellated bodonids, a sister group of trypanosomatids within kinetoplastids, harbor the cytosolic DHOD. Further, Bodo saliens, a bodonid, had an ACT/DHOD gene fusion encoding aspartate carbamoyltransferase (ACT), the second enzyme of the de novo pyrimidine pathway, and DHOD. This is the first report of this novel gene structure. These results are consistent with suggestions that an ancient common ancestor of Euglenozoa had a mitochondrial DHOD whose descendant exists in E. gracilis and that a common ancestor of kinetoplastids (bodonids and trypanosomatids) subsequently acquired a cytosolic DHOD by horizontal gene transfer. The cytosolic DHOD gene thus acquired may have contributed to adaptation to anaerobiosis in the kinetoplastid lineage and further contributed to the subsequent establishment of parasitism in a trypanosomatid ancestor. Different molecular strategies for anaerobic adaptation in pyrimidine biosynthesis, used by kinetoplastids and by euglenoids, are discussed. Evolutionary implications of the ACT/DHOD gene fusion are also discussed.Sequence availability: The nucleotide sequence data reported here appear in the GenBank, EMBL, and DDBJ databases with the accession numbers AB120414, AB159227, and AB159228 for Euglena gracilis dihydroorotate dehydrogenase (DHOD), Bodo saliens aspartate carbamoyltransferase/dihydroorotate dehydrogenase (ACT/DHOD), and B. caudatus DHOD, respectively.Reviewing Editor: Dr. Patrick Keeling     

Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane-associated N-terminal domain

The flavin enzyme dihydroorotate dehydrogenase (DHOD; EC 1.3.99.11) catalyzes the oxidation of dihydroorotate to orotate, the fourth step in the de novo pyrimidine biosynthesis of UMP. The enzyme is a promising target for drug design in different biological and clinical applications for cancer and arthritis. The first crystal structure of the class 2 dihydroorotate dehydrogenase from rat has been determined in complex with its two inhibitors brequinar and atovaquone. These inhibitors have shown promising results as anti-proliferative, immunosuppressive, and antiparasitic agents. A unique feature of the class 2 DHODs is their N-terminal extension, which folds into a separate domain comprising two alpha-helices. This domain serves as the binding site for the two inhibitors and the respiratory quinones acting as the second substrate for the class 2 DHODs. The orientation of the first N-terminal helix is very different in the two complexes of rat DHOD (DHODR). Binding of atovaquone causes a 12 A movement of the first residue in the first alpha-helix. Based on the information from the two structures of DHODR, a model for binding of the quinone and the residues important for the interactions could be defined. His 56 and Arg 136, which are fully conserved in all class 2 DHODs, seem to play a key role in the interaction with the electron acceptor. The differences between the membrane-bound rat DHOD and membrane-associated class 2 DHODs exemplified by the Escherichia coli DHOD has been investigated by GRID computations of the hydrophobic probes predicted to interact with the membrane.     

Enzymes of the orotate biosynthetic pathway in Crithidia fasciculata.

A study of the enzymes of the orotate biosynthetic pathway in the kinetoplasid flagellate Crithidia fasciculata has revealed a number of differences between them and those of other organisms, either prokaryotic or eukaryotic. Carbamyl phosphate synthesis could not be demonstrated in cell-free extracts. However, the incorporation of both CO2 and the ureide carbon of citrulline into pyrimidines occurs in growing cells, the latter predominating over the former. The aspartate transcarbamylase of the flagellate has properties which are similar to those of this enzyme as it occurs in mammals rather than other microorganisms. Two enzymes, dihydroorotate synthetase and dihydroorotate hydrolase, are present, the former being responsible for the conversion of carbamylasparate to dihydroorotate. Dihydroorotate hydroxylase, a soluble enzyme requiring a reduced pteridine as a cofactor, converts dihydroorotate to orotate. The hydroxylase is inhibited by orotate, but not by pyrimidine or purine ribonucleotides. Thus orotate serves to control its own biosynthesis.