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Mosquito has a single multisubstrate deoxyribonucleoside kinase characterized by unique substrate specificity

In mammals four deoxyribonucleoside kinases, with a relatively restricted specificity, catalyze the phosphorylation of the four natural deoxyribonucleosides. When cultured mosquito cells, originating from the malaria vector Anopheles gambiae, were examined for deoxyribonucleoside kinase activities, only a single enzyme was isolated. Subsequently, the corresponding gene was cloned and over-expresse

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

The mitochondrial membrane bound dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) catalyzes the fourth step of pyrimidine biosynthesis. By the present correction of a known cDNA sequence for Arabidopsis thaliana DHODH we revealed the importance of the very C-terminal part for its catalytic activity and the reason why--in contrast to mammalian and insect species--the recombinant plant flavoenzyme

Drosophila melanogaster dihydroorotate dehydrogenase : the N-terminus is important for biological function in vivo but not for catalytic properties in vitro

Dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis, is an integral flavoprotein of the inner mitchondrial membrane and is functionally connected to the respiratory chain. Here, experiments have been directed toward determining the roles of the N-terminal sequence motifs both in enzymatic properties of insect DHODH produced in vitro and the in vivo

A few amino acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines

In mammals, the four native deoxyribonucleosides are phosphorylated to the corresponding monophosphates by four deoxyribonucleoside kinases, which have specialized substrate specificities. These four enzymes are likely to originate from a common progenitor kinase. Insects appear to have only one multisubstrate deoxyribonucleoside kinase (dNK, EC 2.7.1.145), which prefers pyrimidine nucleosides, bu

Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation

In eukaryotic cells deoxyribonucleoside kinases belonging to three phylogenetic sub-families have been found: (i) thymidine kinase 1 (TK1)-like enzymes, which are strictly pyrimidine deoxyribonucleoside-specific kinases; (ii) TK2-like enzymes, which include pyrimidine deoxyribonucleoside kinases and a single multisubstrate kinase from Drosophila melanogaster (Dm-dNK); and (iii) deoxycytidine/deoxy

Structural basis for substrate specificities of cellular deoxyribonucleoside kinases

Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides and activate a number of medically important nucleoside analogs. Here we report the structure of the Drosophila deoxyribonucleoside kinase with deoxycytidine bound at the nucleoside binding site and that of the human deoxyguanosine kinase with ATP at the nucleoside substrate binding site. Compared to the human kinase, the Drosophila ki

Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase. : A versatile tool to rate inhibitor effects?

Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expr

Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster

In contrast to all known deoxyribonucleoside kinases, a single highly efficient deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) is able to phosphorylate all precursor nucleosides for DNA synthesis. Dm-dNK was mutated in vitro by high-frequency random mutagenesis, expressed in the thymidine kinase-deficient Escherichia coli strain KY895 and clones were selected for sensitivity to t

Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase

In animals, dihydroorotate dehydrogenase (DHODH) is a mitochondrial protein that carries out the fourth step in de novo pyrimidine biosynthesis. Because this is the only enzyme of this pathway that is localized to mitochondria and because the enzyme is cytosolic in some bacteria and fungi, we carried out studies to understand the mode of targeting of animal DHODH and its submitochondrial localizat

Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its C-terminal deletion mutants

The occurrence of a deoxyribonucleoside kinase in Drosophila melanogaster (Dm-dNK) with remarkably broad substrate specificity has recently been indicated (Munch-Petersen, B., Piskur, J., and Søndergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). To prove that the capacity to phosphorylate all four deoxyribonucleosides is in fact associated to one polypeptide chain, partially sequenced cDNA clones

Redoxal as a new lead structure for dihydroorotate dehydrogenase inhibitors : a kinetic study of the inhibition mechanism

Mitochondrial dihydroorotate dehydrogenase (DHOdehase; EC 1.3.99.11) is a target of anti-proliferative, immunosuppressive and anti-parasitic agents. Here, redoxal, (2,2'-[3,3'-dimethoxy[1, 1'-biphenyl]-4,4'-diyl)diimino]bis-benzoic acid, was studied with isolated mitochondria and the purified recombinant human and rat enzyme to find out the mode of kinetic interaction with this target. Its pattern

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

Species-related inhibition of human and rat dihydroorotate dehydrogenase by immunosuppressive isoxazol and cinchoninic acid derivatives

The isoxazol leflunomide (N-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide) and its active metabolite A77-1726 (N-(4-trifluoromethyl)-phenyl-2-cyano-3-hydroxy-crotonic acidamide) are promising disease-modifying antirheumatic drugs now in clinical trials. The malononitrilamides MNA279 (2-cyano-3-cyclopropyl-3-oxo-(4-cyanophenyl)propionamide) MNA715(N-(4-trifluoromethyl)-phenyl-2-cyano-3-h

Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase

Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in Trichoplusia ni cells, a procedure is described that allo

Rat dihydroorotate dehydrogenase : isolation of the recombinant enzyme from mitochondria of insect cells

Mammalian dihydroorotate dehydrogenase (EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis is located in the mitochondrial inner membrane with functional connection to the respiratory chain. From the cDNA of rat liver dihydroorotate dehydrogenase cloned in our laboratory the first complete sequence of a mammalian enzyme was deduced. Two hydrophobic stretches centered around residues

Functional expression of a fragment of human dihydroorotate dehydrogenase by means of the baculovirus expression vector system, and kinetic investigation of the purified recombinant enzyme

Human mitochondrial dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) has been overproduced by means of a recombinant baculovirus that contained the human cDNA fragment for this protein. After virus infection and protein expression in Trichoplusia ni cells (BTI-Tn-5B1-4), the subcellular distribution of the recombinant dihydroorotate dehydrogenase was determined by t

Anti-peptide immunoglobulins from rabbit and chicken eggs recognise recombinant human dihydroorotate dehydrogenase and a 44-kDa protein from rat liver mitochondria

Mitochondrially bound dihydroorotate dehydrogenase catalyses the fourth sequential step in the de novo synthesis of uridine monophosphate. 312-bp and 983-bp regions of the human dihydroorotate dehydrogenase sequence (1496 bp) were amplified by the polymerase chain reaction, and subcloned into the expression vector pQE 32. The identity of the PCR products was verified by dideoxynucleotide sequencin

'Like Locusts on the Mississippi' : A Pioneer Study of the Expansion of the Swedish Public Sector

In 1962, the first thorough investigation into the expansion of the Swedish public sector was presented as a doctoral dissertation by Erik Höök. This article investigates the origin and reception of this thesis. Höök’s work has been consid ered a standard work of financial statistics. However, it attracted heavy criticism in the media and from other academics for having explained the public sector

Education and health : Long-run effects of peers, tracking and years

We investigate two parallel school reforms in Sweden to assess the long-run health effects of education. One reform only increased years of schooling, while the other increased years of schooling but also removed tracking leading to a more mixed socioeconomic peer group. By differencing the effects of the parallel reforms we separate the effect of de-tracking and peers from that of more schooling.

Fragmentation, commodification and responsibilisation in the governing of flood risk mitigation in Sweden

The purpose of this paper is to increase our understanding of the governing of flood risk mitigation in advanced liberal society, through an in-depth Swedish case study. By combining social network analysis and genealogy, this paper investigates who is involved, how they organise, their modes of thinking, how they mitigate flood risk, as well as how such regime of practises have come into being. T