Two different dihydroorotate dehydrogenases in Lactococcus lactis.

The pyrimidine de novo biosynthesis pathway has been characterized for a number of organisms. The general pathway consists of six enzymatic steps. In the characterization of the pyrimidine pathway of Lactococcus lactis, two different pyrD genes encoding dihydroorotate dehydrogenase were isolated. The nucleotide sequences of the two genes, pyrDa and pyrDb, have been determined. One of the deduced amino acid sequences has a high degree of homology to the Saccharomyces cerevisiae dihydroorotate dehydrogenase, and the other resembles the dihydroorotate dehydrogenase from Bacillus subtilis. It is possible to distinguish between the two enzymes in crude extracts by using different electron acceptors. We constructed mutants containing a mutated form of either one or the other or both of the pyrD genes. Only the double mutant is pyrimidine auxotrophic.     

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.     

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     

Functional expression of human dihydroorotate dehydrogenase (DHODH) in pyr4 mutants of ustilago maydis allows target validation of DHODH inhibitors in vivo

Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.     

A new type of dihydroorotate dehydrogenase,type 1S,from the thermoacidophilic archaeon<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>

Dihydroorotate dehydrogenase (DHOD) (EC 1.3.3.1) from the thermoacidophilic archaeon Sulfolobus solfataricus P2 (DSM 1617) was partially purified 3,158-fold, characterized, and the encoding genes identified. Based on enzymological as well as phylogenetic methods, dihydroorotate dehydrogenase from S. solfataricus (DHODS) represents a new type of DHOD, type 1S. Furthermore, it is unable to use any of the (type-specific) natural electron acceptors employed by all other presently known DHODs. DHODS shows optimal activity at 70 degrees C in the pH range 7-8.5. It is capable of using ferricyanide, 2,6-dichlorophenolindophenol (DCIP), Q(0), and molecular oxygen as electron acceptor. Kinetic studies employing ferricyanide indicate a two-site ping-pong mechanism with K(M) values of 44.2+/-1.9 microM for the substrate dihydroorotate and 344+/-21 microM for the electron acceptor ferricyanide, as well as competitive product inhibition with a K(i) of 23.7+/-3.4 microM for the product orotate (OA). The specific activity, as determined from a partially purified sample, is approximately 20 micromol mg(-1) min(-1). DHODS is a heteromeric enzyme comprising a catalytic subunit encoded by pyrD (291 aa; MW=31.1 kDa) and an electron acceptor subunit (208 aa; MW=23.6 kDa), encoded by orf1. DHODS employs a serine as catalytic base, which is unique for a cytosolic DHOD. To our knowledge, this work represents not only the first study on an archaeal DHOD but the first on a nonmesophilic DHOD as well.     

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。     

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.     

Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.     

Kinetics of inhibition of human and rat dihydroorotate dehydrogenase by atovaquone, lawsone derivatives, brequinar sodium and polyporic acid

Mitochondrially-bound dihydroorotate dehydrogenase (EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. The enzyme has been identified as or surmised to be the pharmacological target for isoxazol, triazine, cinchoninic acid and (naphtho)quinone derivatives, which exerted antiproliferative, immunosuppressive, and antiparasitic effects. Despite this broad spectrum of biological and clinical relevance, there have been no comparative studies on drug-dihydroorotate dehydrogenase interactions. Here, we describe a study of the inhibition of the purified recombinant human and rat dihydroorotate dehydrogenase by ten compounds. 1,4-Naphthoquinone, 5,8-hydroxy-naphthoquinone and the natural compounds juglon, plumbagin and polyporic acid (quinone derivative) were found to function as alternative electron acceptors with 10-30% of control enzyme activity. The human and rat enzyme activity was decreased by 50% by the natural compound lawsone ( > 500 and 49 microM, respectively) and by the derivatives dichloroally-lawsone (67 and 10 nM), lapachol (618 and 61 nM) and atovaquone (15 microM and 698 nM). With respect to the quinone co-substrate of the dihydroorotate dehydrogenase, atovaquone (Kic = 2.7 microM) and dichloroally-lawsone (Kic = 9.8 nM) were shown to be competitive inhibitors of human dihydroorotate dehydrogenase. Atovaquone (Kic = 60 nM) was also acompetitive inhibitor of the rat enzyme. Dichloroally]-lawsone was found to be a time-dependent inhibitor of the rat enzyme, with the lowest inhibition constant (Ki* = 0.77 nM) determined so far for mammalian dihydroorotate dehydrogenases. Another inhibitor, brequinar was previously reported to be a slow-binding inhibitor of the human dihydroorotate dehydrogenase [W. Knecht, M. Loffler, Species-related inhibition of human and rat dihyroorotate dehydrogenase by immunosuppressive isoxazol and cinchoninic acid derivatives, Biochem. Pharmacol. 56 (1998) 1259-1264]. The slow binding features of this potent inhibitor (Ki* = 1.8 nM) with the human enzyme, were verified and seen to be one of the reasons for the narrow therapeutic window (efficacy versus toxicity) reported from clinical trials on its antiproliferative and immunosuppressive action. With respect to the substrate dihydroorotate, atovaquone was an uncompetitive inhibitor of human dihydroorotate dehydrogenase (Kiu = 11.6 microM) and a non-competitive inhibitor of the rat enzyme (Kiu = 905/ Kic = 1,012 nM). 1.5 mM polyporic acid, a natural quinone from fungi, influenced the activity of the human enzyme only slightly; the activity of the rat enzyme was decreased by 30%.     

Catalytic enzyme histochemistry and biochemical analysis of dihydroorotate dehydrogenase/oxidase and succinate dehydrogenase in mammalian tissues,cells and mitochondria

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimate electron transfer system, whereas the rest of pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the nitroblue tetrazolium technique. In addition, a hydrogen peroxide-producing oxidase side-reactivity of dihydroorotate dehydrogenase could be visualized by trapping the peroxide with cerium-diaminobenzidine. The pattern of activity was similar to that of succinate dehydrogenase, but revealed a less intensive staining. High activities of dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastro-intestinal tract, cultured Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle. Dihydroorotate dehydrogenase and succinate dehydrogenase activity in Ehrlich ascites tumor cells grown in suspension culture were quantified by application of nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble formazans with organic solvents. The ratio of dihydroorotate dehydrogenase to succinate dehydrogenase activity was 14. This was in accordance with that of 15 obtained from oxygen consumption measurement of isolated mitochondria on addition of dihydroorotate or succinate. The ratio determined with mitochondria from animal tissues was up to 115 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.     

Identification, isolation, and overexpression of the gene encoding the psi subunit of DNA polymerase III holoenzyme.

The gene encoding the psi subunit of DNA polymerase III holoenzyme, holD, was identified and isolated by an approach in which peptide sequence data were used to obtain a DNA hybridization probe. The gene, which maps to 99.3 centisomes, was sequenced and found to be identical to a previously uncharacterized open reading frame that overlaps the 5' end of rimI by 29 bases, contains 411 bp, and is predicted to encode a protein of 15,174 Da. When expressed in a plasmid that also expressed holC, holD directed expression of the psi subunit to about 3% of total soluble protein.     

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.     

Isolation and characterization of the moxJ, moxG, moxI, and moxR genes of Paracoccus denitrificans: inactivation of moxJ, moxG, and moxR and the resultant effect on methylotrophic growth.

By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the 5' part of a sixth one. The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, moxG, and moxI gene products of Methylobacterium extorquens AM1. Directly downstream from these three genes, a new mox gene was identified. The gene is designated moxR. By using the suicide vector pGRPd1, the moxJ, moxG, and moxR genes were inactivated by the insertion of a kanamycin resistance gene. Subsequently, suicide vector pRVS1 was used to replace the marker genes in moxJ and moxG for unmarked deletions made in vitro. As a result, the three insertion strains as well as the two unmarked mutant strains were unable to grow on methanol, even in the presence of pyrroloquinoline quinone. Growth on succinate and on methylamine was not affected. In all five mutant strains, synthesis of the large subunit of methanol dehydrogenase and of inducible cytochrome c553i was observed. The moxJ and moxG insertion mutant strains were unable to synthesize both the cytochrome c551i and the small subunit of methanol dehydrogenase, and this lack of synthesis was attended by the loss of methanol dehydrogenase activity. The moxJ deletion mutant strain partly synthesized the latter two proteins, cytochrome c551i. Partial synthesis of the small subunit of methanol dehydrogenase observed with the latter strain was attended by a corresponding extent of methanol dehydrogenase activity. The moxR insertion mutant strain was shown to synthesize cytochrome c551i as well as the large and small subunits of methanol dehydrogenase, but no methanol dehydrogenase activity was observed. The results show that periplasmic cytochrome c551i is the moxG gene product and the natural electron acceptor of methanol dehydrogenase in P. denitrificans. In contrast to earlier suggestions, this cytochrome was found to be different from membrane-bound cytochrome c552. In addition, it is demonstrated that moxI encodes the small subunit of methanol dehydrogenase. It is suggested that MoxJ is involved in the assemblage of active methanol dehydrogenase in the periplasm and, in addition, that MoxR is involved in the regulation of formation of active methanol dehydrogenase.     

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.     

Lys-D48 is required for charge stabilization, rapid flavin reduction, and internal electron transfer in the catalytic cycle of dihydroorotate dehydrogenase B of Lactococcus lactis

Dihydroorotate dehydrogenase B (DHODB) catalyzes the oxidation of dihydroorotate (DHO) to orotate and is found in the pyrimidine biosynthetic pathway. The Lactococcus lactis enzyme is a dimer of heterodimers containing FMN, FAD, and a 2Fe-2S center. Lys-D48 is found in the catalytic subunit and its side-chain adopts different positions, influenced by ligand binding. Based on crystal structures of DHODB in the presence and absence of orotate, we hypothesized that Lys-D48 has a role in facilitating electron transfer in DHODB, specifically in stabilizing negative charge in the reduced FMN isoalloxazine ring. We show that mutagenesis of Lys-D48 to an alanine, arginine, glutamine, or glutamate residue (mutants K38A, K48R, K48Q, and K48E) impairs catalytic turnover substantially (approximately 50-500-fold reduction in turnover number). Stopped-flow studies demonstrate that loss of catalytic activity is attributed to poor rates of FMN reduction by substrate. Mutation also impairs electron transfer from the 2Fe-2S center to FMN. Addition of methylamine leads to partial rescue of flavin reduction activity. Nicotinamide coenzyme oxidation and reduction at the distal FAD site is unaffected by the mutations. Formation of the spin-interacting state between the FMN semiquinone-reduced 2Fe-2S centers observed in wild-type enzyme is retained in the mutant proteins, consistent with there being little perturbation of the superexchange paths that contribute to the efficiency of electron transfer between these cofactors. Our data suggest a key charge-stabilizing role for Lys-D48 during reduction of FMN by dihydroorotate, or by electron transfer from the 2Fe-2S center, and establish a common mechanism of FMN reduction in the single FMN-containing A-type and the complex multicenter B-type DHOD enzymes.     

Dihydroorotate dehydrogenase mRNA and protein expression analysis in normal and drug-resistant cells

To follow the expression of the fourth enzyme of pyrimidine de novo synthesis dihydroorotate dehydrogenase (DHODH) in cells and tissues, we studied the DHODH mRNA expression by means of RT-PCR in rat tissues. Rabbit polyclonal anti-DHODH immunoglobulins were applied for immunochemical quantification of the enzyme protein by Western blotting. In mouse B-lymphocytes, which were adapted to tolerate up to a 50-fold concentration of the DHODH inhibitor leflunomide, a 20 fold protein overexpression was measured. Southern blotting indicated DHODH gene amplification.