Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The B form of dihydroorotate dehydrogenase from Lactococcus lactis (DHOdehase B) is encoded by the pyrDb gene. However, recent genetic evidence has revealed that a co-transcribed gene, pyrK, is needed to achieve the proper physiological function of the enzyme. We have purified DHOdehase B from two strains of Escherichia coli, which harbored either the pyrDb gene or both the pyrDb and the pyrK genes of L. lactis on multicopy plasmids. The enzyme encoded by pyrDb alone (herein called the delta-enzyme) was a bright yellow, dimeric protein that contained one molecule of tightly bound FMN per subunit. The delta-enzyme exhibited dihydroorotate dehydrogenase activity with dichloroindophenol, potassium hexacyanoferrate(III), and molecular oxygen as electron acceptors but could not use NAD+. The DHOdehase B purified from the E. coli strain that carried both the pyrDb and pyrK genes on a multicopy plasmid (herein called the deltakappa-enzyme) was quite different, since it was formed as a complex of equal amounts of the two polypeptides, i.e. two PyrDB and two PyrK subunits. The deltakappa-enzyme was orange-brown and contained 2 mol of FAD, 2 mol of FMN, and 2 mol of [2Fe-2S] redox clusters per mol of native protein as tightly bound prosthetic groups. The deltakappa-enzyme was able to use NAD+ as well as dichloroindophenol, potassium hexacyanoferrate(III), and to some extent molecular oxygen as electron acceptors for the conversion of dihydroorotate to orotate, and it was a considerably more efficient catalyst than the purified delta-enzyme. Based on these results and on analysis of published sequences, we propose that the architecture of the deltakappa-enzyme is representative for the dihydroorotate dehydrogenases from Gram-positive bacteria.
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PMID:The B form of dihydroorotate dehydrogenase from Lactococcus lactis consists of two different subunits, encoded by the pyrDb and pyrK genes, and contains FMN, FAD, and [FeS] redox centers. 891 May 99

Enterococcus faecalis dihydroorotate dehydrogenase B is a heterodimer of 28 and 33 kDa encoded by the pyrK and pyrDb genes. Both subunits copurify during all chromatographic steps, and, as determined by HPLC, one FMN and one FAD are bound per heterodimer. The enzyme catalyzes efficient oxidation of 4-S-NADH by orotate. Isotope effect and pH data suggest that reduction of flavin by NADH at the PyrK site is only partially rate limiting with no kinetically significant proton transfer occurring in the reductive half-reaction; therefore, a group exhibiting a pK of 5.7 +/- 0.2 represents a residue involved in binding of NADH rather than in catalysis. The reducing equivalents are shuttled between the NADH-oxidizing flavin in PyrK and the orotate-reacting flavin in PyrDb, by iron-sulfur centers through flavin semiquinones as intermediates. A solvent kinetic isotope effect of 2.5 +/- 0.2 on V is indicative of rate-limiting protonation in the oxidative half-reaction and most likely reflects the interaction between the isoalloxazine N1 of the orotate-reducing flavin and Lys 168 (by analogy with L. lactis DHODase A). The oxidative half-reaction is facilitated by deprotonation of the group(s) with pK(s) of 5.8-6.3 and reflects either deprotonation of the reduced flavin or binding of orotate; this step is followed by hydride transfer to C6 and general acid-assisted protonation (pK of 9.1 +/- 0.2) at C5 of the product.
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PMID:Dihydroorotate dehydrogenase B of Enterococcus faecalis. Characterization and insights into chemical mechanism. 1052 84

Bacillus subtilis dihydroorotate dehydrogenase (DHOD) consists of two subunits, PyrDI (M(r) = 33,094) and PyrDII (M(r) = 28,099). The two subunits were overexpressed jointly and individually and purified. PyrDI was an FMN-containing flavoprotein with an apparent native molecular mass of 85,000. Overexpressed PyrDII formed inclusion bodies and was purified by refolding and reconstitution. Refolded PyrDII bound 1 mol FAD and 1 mol [2Fe-2S] per mol PyrDII. Coexpression and purification of PyrDI and PyrDII yielded a native holoenzyme complex with an apparent native molecular mass of 114,000 that indicated a heterotetramer (PyrDI(2)PyrDII(2)). The holoenzyme possessed dihydroorotate:NAD(+) oxidoreductase activity and could also reduce menadione and artificial dyes. Purified PyrDI also possessed DHOD activity but could not reduce NAD(+). Compared to PyrDI, the holoenzyme had a more than 20-fold smaller K(m) value for dihydroorotate, an approximately 50-fold smaller K(i) value for orotate, and approximately 500-fold greater catalytic efficiency. Dihydroorotate:NAD(+) oxidoreductase activity could be recovered by mixing the purified subunits. Recovered activity showed a clear dependence on FAD reconstitution of PyrDII but not on reconstitution with FeS clusters. PyrDII had a strong preference for FAD over FMN and bound it with an estimated K(d) value of 4.9 +/- 0.8 nM. pyrDII mutants containing alanine substitutions of the cysteine ligands to the [2Fe-2S] cluster failed to complement the pyr bradytrophy of a DeltapyrDII strain, indicating a requirement for the FeS cluster in PyrDII for normal function in vivo.
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PMID:Biochemical characterization of the heteromeric Bacillus subtilis dihydroorotate dehydrogenase and its isolated subunits. 1054 5

The mitochondrial electron transport chain (ETC) is necessary for tumour growth^1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies^7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria^10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)¹², which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)¹³ targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.
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PMID:Mitochondrial ubiquinol oxidation is necessary for tumour growth. 3264 34

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