Plant dihydroorotate dehydrogenase differs significantly in substrate specificity and inhibition from the animal enzymes

Ullrich, Alexandra; Knecht, Wolfgang; Piškur, Jure; Löffler, Monika

doi:10.1016/s0014-5793(02)03425-7

Cited by 38 publications

(30 citation statements)

References 32 publications

(68 reference statements)

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“…Both proteins are not or are only weakly annotated as organellar by corresponding prediction software (Schwacke et al, 2003), supporting these results. DHODH locates to mitochondria based on GFP fusion protein analysis (Figure 8) in good agreement with previously reported data (Ullrich et al, 2002;Zrenner et al, 2006). Based on these findings, we propose the following hypothesis: Pyrimidine de novo synthesis in Arabidopsis is located in the plastid, mitochondrion, and cytosol.…”

Section: Plastid Pyrimidine Metabolism Depends On Salvagesupporting

confidence: 91%

De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis

et al. 2012

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Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 mM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

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Section: Plastid Pyrimidine Metabolism Depends On Salvagesupporting

confidence: 91%

De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis

et al. 2012

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“…Since DCIP can accept electrons in the absence of ubiquinone, this background activity was measured (k cat = 28.1 ± 0.7 sec −1 ) and was subtracted from activities in the presence of the ubiquinones shown in Table 1. The activities of quinones at 0.1 mM, such as PQ 0 , Q 10 , and menadione [35, 36], were measured and compared to the activity in the presence of Q D .…”

Section: Methodsmentioning

confidence: 99%

“…Q 10 presented solubility problems; to prevent precipitation the ethanol concentration in the reaction assay was raised to 10%, a concentration that did not affect TgDHOD activity, as is the case for other DHODs [36]. …”

Section: Methodsmentioning

confidence: 99%

Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii

Triana

Huynh

Garavito

et al. 2012

Molecular and Biochemical Parasitology

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Summary The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84 U/mg, a kcat of 89 sec−1, a Km = 60 μM for L-dihydroorotate, and a Km = 29 μM for decylubiquinone (QD). Quinones lacking or having short isoprenoid side chains yielded lower kcats than QD. As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The The IC50s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91 μM, 96 μM, and 60 μM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite’s mitochondrion, revealing this organelle as the site of orotate production in T gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs.

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“…In the case of plant DHODH, nothing is known about posttranscriptional regulation. The plant enzyme has only recently become accessible for in vitro characterization, and a significant structural difference to the animal enzymes has been reported (Ullrich et al, 2002). A suitable antibody was not available to further characterize the reduction on protein level.…”

Section: Reduced Steady-state Mrna Levels Do Not Necessarily Lead To mentioning

confidence: 99%

Functional Analysis of the Pyrimidine de Novo Synthesis Pathway in Solanaceous Species

2005

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Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.Pyrimidine nucleotides are abundant molecules with essential functions in a multitude of biochemical processes. They are of particular importance in dividing and elongating tissues as building blocks for nucleic acid biosynthesis. In addition, as an energy source or precursors for the synthesis of primary and secondary products, they are participants in various metabolic processes. In particular, the pyrimidine nucleotides are directly involved in plant carbohydrate metabolism providing the energy-rich precursor UDP-Glc for many synthetic reactions, such as Suc and cell wall biosyntheses.In recent years, the basic processes of plant nucleotide de novo synthesis have been analyzed in some detail (Giermann et al., 2002;Moffatt and Ashihara, 2002;Boldt and Zrenner, 2003;Stasolla et al., 2003;Kafer et al., 2004). Plants utilize the same reactions as those found in other organisms. The so-called orotate pathway of pyrimidine de novo synthesis is defined as the formation of UMP from carbamoyl phosphate, Asp, and phosphoribosyl pyrophosphate. The pathway consists of the six enzymatic reactions as shown in Figure 1. The first reaction is conducted by the Gln hydrolyzing carbamoyl phosphate synthase. This enzyme is not unique to the pyrimidine pathway but is also involved in Arg biosynthesis. It is composed of two different subunits (carbamoyl phosp...

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Plant dihydroorotate dehydrogenase differs significantly in substrate specificity and inhibition from the animal enzymes

Cited by 38 publications

References 32 publications

De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis

De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis

Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii

Functional Analysis of the Pyrimidine de Novo Synthesis Pathway in Solanaceous Species

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