Dihydroorotate dehydrogenase from<i>Saccharomyces cerevisiae</i>: spectroscopic investigations with the recombinant enzyme throw light on catalytic properties and metabolism of fumarate analogues

Zameitat, Elke; Pierik, Antonio J.; Zocher, Kathleen; Löffler, Monika

doi:10.1111/j.1567-1364.2007.00275.x

Cited by 20 publications

(19 citation statements)

References 36 publications

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“…A large red-shift is observed upon OA binding to oxidized Class 1A and Class 2 DHODs (14, 18–21). The maximum flavin peak in the E. coli DHOD shifts from 456 nm to 475 nm.…”

Section: Resultsmentioning

confidence: 99%

Roles in Binding and Chemistry for Conserved Active Site Residues in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli

Fagan

Palfey

2009

Biochemistry

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Dihydroorotate dehydrogenases (DHODs) catalyze the only redox step in de novo pyrimidine biosynthesis, the oxidation of dihydroorotate (DHO) to orotate (OA). During the reaction, the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of an enzyme-bound FMN prosthetic group as a hydride and an active site base (Ser175 in the Class 2 DHOD from E. coli) deprotonates C5 of DHO. Aside from the identity of the active site base, the pyrimidine binding site of all DHODs is nearly identical. Several strictly conserved residues (four asparagines and either a serine or threonine) make extensive hydrogen-bonds to the pyrimidine). The roles these conserved residues play in DHO oxidation is unknown. Site-directed mutagenesis was used to investigate the role of each residue during DHO oxidation. The effects of each mutation on substrate and product binding, as well as the effect on the rate constant of the chemical step were determined. The effects of the mutations ranged from negligible to severe. Some of the residues are very important for chemistry, while others were important for binding. Mutation of residues capable of stabilizing reaction intermediates resulted in large decreases in the rate constant of the chemical step, suggesting these residues are quite important for stabilizing charge build-up in the active site. This finding is consistent with previous results that Class 2 DHODs use a stepwise mechanism for DHO oxidation.Dihydroorotate dehydrogenases (DHODs 1 ) are flavin-containing enzymes that catalyze the fourth step (the only redox step) in the de novo synthesis of pyrimidines -the conversion of dihydroorotate (DHO) to orotate (OA) (Scheme 1). DHODs have been categorized into two broad classes based on sequence (1). Several properties of the enzymes segregate nicely into these classes. Class 2 DHODs are membrane-bound enzymes that are oxidized by ubiquinone (2). Class 1 enzymes are cytosolic proteins that have been further divided into two subclasses. Class 1A enzymes are homodimers that are oxidized by fumarate (3). Class 1B enzymes are α 2 b 2 heterotetramers that contain a subunit that resembles the Class 1A enzymes and a second subunit that contains FAD and an iron-sulfur cluster, allowing the enzyme to be oxidized by NAD (4). Class 1 DHODs are found mostly in Gram-positive bacteria, although a few microbial eukaryotes also have Class 1A DHODs, while Class 2 enzymes are found in Gram-negative bacteria and almost all eukaryotes.*To whom correspondence should be addressed at: Department of Biological Chemistry, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, Phone: (734) 615-2452; Fax: (734) The catalytic cycle of DHODs can be studied in two separate half-reactions, allowing the direct observation of the chemistry. The reductive half-reaction involves the oxidation of DHO to OA with the concomitant reduction of the enzyme-bound FMN. The kinetics of the reductive half-reaction has been studied in anaerobic stopped-flow experiments for several Class 2 ...

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“…A large red-shift is observed upon OA binding to oxidized Class 1A and Class 2 DHODs (14, 18–21). The maximum flavin peak in the E. coli DHOD shifts from 456 nm to 475 nm.…”

Section: Resultsmentioning

confidence: 99%

Roles in Binding and Chemistry for Conserved Active Site Residues in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli

Fagan

Palfey

2009

Biochemistry

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“…Altogether, flavoproteins of β-oxidation account for 80% of the total content of mitochondrial flavoenzymes [50]. (iii) Compared to other complexes in the respiratory chain (complex I [51,52], complex II [53], glycerol 3-phosphate dehydrogenase [54,55], and dihydroorotate dehydrogenase [56,57]), ETFQOR has the most positive redox potentials (+27 mV for FAD 1e- /FAD ox and +47 mV for [FeS] 2+ /[FeS] 1+ ) [58,59]. Therefore it is thermodynamically favorable for a reduced Q pool to reduce ETFQOR and possibly ETF.…”

Section: Discussionmentioning

confidence: 99%

Sites of superoxide and hydrogen peroxide production during fatty acid oxidation in rat skeletal muscle mitochondria

Perevoshchikova

Quinlan

Orr

et al. 2013

Free Radical Biology and Medicine

110

102

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H2O2 production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined under two conditions: the absence of respiratory chain inhibitors and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H2O2 production were low unless carnitine or malate was added to limit acetyl-CoA accumulation. With palmitoylcarnitine alone, H2O2 production was dominated by complex II (44% from site IIF in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site IF). With added carnitine, H2O2 production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site IIIQo) and 25% from site IF. Thus complex II (site IIF in the forward reaction) is a major source of H2O2 production during oxidation of palmitoylcarnitine ± carnitine. Under the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H2O2 production were highest in the presence of palmitoylcarnitine ± carnitine and were dominated by complex II (site IIF in the reverse reaction). About half the rest was from site IF, but a significant portion, ~40 pmol H2O2 · min−1 · mg protein−1, was not from complex I, II, or III and was attributed to the proteins of β-oxidation (electron-transferring flavoprotein (ETF) and ETF-ubiquinone oxidoreductase). The maximum rate from the ETF system was ~200 pmol H2O2 · min−1 ~ mg protein−1 under conditions of compromised antioxidant defense and reduced ubiqui-none pool. Thus complex II and the ETF system both contribute to H2O2 production during fatty acid oxidation under appropriate conditions.

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“…; Zameitat et al. ). This unique property explains why both glucose‐grown yeast and some trypanosomes, when they utilize sugars as their sole source of energy, are capable of suppressing their respiratory chain without affecting their pyrimidine biosynthesis.…”

Section: Observations and Predictionsmentioning

confidence: 98%

“…Kinetoplastids lack mitochondrial DHODH, which has been replaced by cytosolic isofunctional enzyme of the bacterial type that uses fumarate as electron acceptor. This situation is unique among eukaryotes and has so far been encountered only in these flagellates and yeast (Annoura et al 2005;Zameitat et al 2007). This unique property explains why both glucose-grown yeast and some trypanosomes, when they utilize sugars as their sole source of energy, are capable of suppressing their respiratory chain without affecting their pyrimidine biosynthesis.…”

Section: Mitochondrionmentioning

confidence: 99%

Comparative Metabolism of Free‐living Bodo saltans and Parasitic Trypanosomatids

Opperdoes

Butenko

Flegontov

et al. 2016

J Eukaryotic Microbiology

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Comparison of the genomes of free-living Bodo saltans and those of parasitic trypanosomatids reveals that the transition from a free-living to a parasitic life style has resulted in the loss of approximately 50% of protein-coding genes. Despite this dramatic reduction in genome size, B. saltans and trypanosomatids still share a significant number of common metabolic traits: glycosomes; a unique set of the pyrimidine biosynthetic pathway genes; an ATP-PFK which is homologous to the bacterial PPi -PFKs rather than to the canonical eukaryotic ATP-PFKs; an alternative oxidase; three phosphoglycerate kinases and two GAPDH isoenzymes; a pyruvate kinase regulated by fructose-2,6-bisphosphate; trypanothione as a substitute for glutathione; synthesis of fatty acids via a unique set of elongase enzymes; and a mitochondrial acetate:succinate coenzyme A transferase. B. saltans has lost the capacity to synthesize ubiquinone. Among genes that are present in B. saltans and lost in all trypanosomatids are those involved in the degradation of mureine, tryptophan and lysine. Novel acquisitions of trypanosomatids are components of pentose sugar metabolism, pteridine reductase and bromodomain-factor proteins. In addition, only the subfamily Leishmaniinae has acquired a gene for catalase and the capacity to convert diaminopimelic acid to lysine.

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Dihydroorotate dehydrogenase fromSaccharomyces cerevisiae: spectroscopic investigations with the recombinant enzyme throw light on catalytic properties and metabolism of fumarate analogues

Cited by 20 publications

References 36 publications

Roles in Binding and Chemistry for Conserved Active Site Residues in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli

Roles in Binding and Chemistry for Conserved Active Site Residues in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli

Sites of superoxide and hydrogen peroxide production during fatty acid oxidation in rat skeletal muscle mitochondria

Comparative Metabolism of Free‐living Bodo saltans and Parasitic Trypanosomatids

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