Dihydro-orotic acid

Lieberman 1, A Komberg (1954) Enzymatic synthesis and breakdown of a pyrimidine, orotic acid II. Dihydro-orotic acid, ureidosuccinic acid, and 5-carboxymethylhydantoin. J Biol Chem 207 911-924. 

L- and DL-dihydro-orotic acid supported growth of Lactobacillus bulgaricus 09. The D-isomer alone is not only without activity, but is reported to reversibly inhibit the growth-promoting property exerted by ureidosuccinic or orotic acid [74, 91]. The dihydro-orotic acid prepared by fusion of maleic acid and urea [130] is inactive in bothi. bulgaricus (B [128] and an enzyme system [72, 131]. A comparison of the urea fusion product and that prepared by catalytic hydrogenation of orotic acid revealed that the former is actually fumarylurea [132]. 

Isoindoline li/-Isoindole, 2,3-dihydro- Orotic acid 4-Pyrimidinecarboxylicacid,l,2,3,6-tetrahydro-2,6-... 

Leflunomide Inhibits dihydro-orotic acid dehydrogenase (DHOD) —4 ump —4 ribonucleotides -> arrests lymphocytes in Gj Alopecia, rash, diarrhea, hepatotoxicity... 

B. leflunomide inhibition of dihydro-orotic acid dehydrogenase... 

Answer B. Leflunomide, used in rheumatoid arthritis, inhibits dihydro-orotic acid dehydrogenase >4 formation of UMP ->4- de novo synthesis of ribonucleotides — arrest of lymphocytes in the GI phase. Glucocorticoids do not decrease expression of lipoxygenase, but by preventing arachidonate formation they decrease activity of the pathway. Misoprostol, used in NSAID-induced GI ulcers, activates receptors colchicine decreases microtubular polymerization ketorolac is a potent NSAID but a nonselective inhibitor of cyclooxygenases. 

LEF inhibits the mitochondrial enzyme dihydro-orotic acid dehydrogenase, which plays a key role in pyrimidine synthesis (129,130). Because it is complimentary to the purine antagonism seen with MTX, these two drugs can be complimentary in action (131). LEF has no reported effect on adenosine. 

The carbamyl group of citrulline can also be transferred to L-aspartate, it is believed through carbamyl phosphate as an intermediate, to form carbamyl-L-asparate (16-18). This in turn is the precursor of dihydro-orotic acid and ultimately of the pyrimidines. 

By ring closure carbamylaspartic acid was transformed into dihydro-orotic acid (Fig. 21). The reaction was freely reversible and the enzyme responsible for this conversion was named dihydroorotase. The enzyme has been found in several bacteria grown in the presence of orotic acid 369,370), in rat liver 371), and in extracts of E. coli 372). The compound originally prepared as dihydroorotic acid 373) was not identical with the enzymic product 374)- However, the authenticity of the enzymic product was proven by several synthetic methods 371, 375). 

The first step of the biosynthesis of pyrimidine nucleotides is the irreversible carbamylation of L-aspartate by carbamyl-phosphate to form carbamylaspartate (catalyzed by the enzyme aspartate transcarbamylase). Next, carbamylaspartate is converted, by ring closure, to dihydro-orotic acid which, in turn, is reduced to orotic acid, catalyzed by the enzyme orotic acid dehydrogenase (OAD). Orotic acid (6-carboxyuracil) reacts with 5 -phosphoribosyl--1-pyrophosphate (PRPP) to form orotidine monophosphate (OMP). 

The Biosynthesis of the Pyrimidine Ring begins with aspartic acid and carbamyl phosphate. The latter is an energy-rich compound which reacts with the former to give carbamylaspartic acid. Ring closure consumes ATP and is in principle an acid amide formation (peptide synthesis). The intermediate dihydro-orotic acid is dehydrogenated to orotic acid, probably by action of a flavoprotein. Orotic acid is the key precursor of pyrimidine nucleotides. It reacts with phosphoribosyl pyrophosphate. The removal of pyrophosphate yields the nucleotide of orotic acid, whose enzymic decarboxylation produces uridine 5 -phosphate. Phosphorylation with ATP yields uridine pyrophosphate and, finally, uridine triphosphate. Beside the above pathway, there is the further possibility of converting free uracil and ribose 1-phosphate to the nucleoside and from there with ATP to the nucleotide. 

Hayon447 to absorb in this UV region. Similar measurements have been made for a number derivatives of uracil (5,6-dihydro, 1-, 3-, and 6-methyl, 1,3-dimethyl-, and 5-aminouracil, orotic acid, barbituric and isobarbituric acids also see similar measurements448 on 5-bromouracil and its iV-methyl derivatives), thymine itself and its derivatives (5,6-dihydro, 2-methylthymine). In the case of cytosine, Hayon447 has very tentatively suggested that species 47 and 48 are obtained on pulse radiolysis of the solution at pH 5.5, and 49 at pH 13.3. 

Brequinar (DUP 785, NSC 368390) is a quinoline carboxylic acid derivative that inhibits pyrimidine synthesis by inhibiting dihydro-orotate dehydrogenase. It was originally developed as an anticancer drug, but has also been investigated for its immunosuppressant activity after transplantation. Some data suggest that that the immunosuppressant activity of brequinar may be partly due to inhibition of tyrosine phosphorylation in lymphocytes (1). 

Dihydroorotase carbamoylaspartic acid amidohydrolase l-5, 6-dihydro-orotate 3.5.2.3... 

The first three reactions are catalyzed by a trifunctional protein which contains carbamoyl-phosphate synthetase II, aspartate carbamoyltransferase and dihydro-orotase. This set of reactions begins with the synthesis of carbamoyl phosphate followed by its condensation with aspartic acid. The third step involves the closure of the ring through the removal of water by the action of dihydro-orotase to yield dihydro-orotate. The fourth enzyme, dihydro-orotate oxidase, oxidizes dihydro-orotate to orotate and is a mitochondrial flavoprotein enzyme located on the outer surface of the inner membrane and utilizes NAD" " as the electron acceptor. The synthesis of UMP from orotate is catalyzed by a bifunctional protein which comprises orotate PRTase and orotidine 5 -phosphate (OMP) decarboxylase. The former phosphoribosylates orotate to give OMP the latter decarboxylates OMP to UMP, the immediate precursor for the other pyrimidine nucleotides. It is interesting to note that whereas five molecules of ATP (including the ATP used in the synthesis of PRPP) are used in the de novo synthesis of IMP, no net ATP is used in the de novo synthesis of UMP. In de novo pyrimidine synthesis, two ATP molecules are used to synthesize carbamoyl phosphate and one ATP is needed to synthesize the PRPP used by orotate PRTase but 3 ATPs... 

The conversion of carbamoyl aspartate to orotic acid became evident in work by Lieberman and Kornberg [97] who studied the degradation of orotic acid by an orotate-fermenting bacterium, Zymobacterium oroticum, and isolated two intermediates, dihydro-orotate and carbamoyl aspartate. The degradation reactions were found to be reversible and the cell-free extract of Z. oroticum converted carbamoyl aspartate back to orotic acid. 

The enzyme catalysing the reversible cyclization of carbamoyl aspartate to dihydro-orotate is called dihydro-orotase (L-4,5-dihydro-orotate amino-hydrolase, EC 3.5.2.3). Dihydro-orotase was found in various animal tissues and for the catalytic function requires Zn + ions [98]. Orotic acid was found to be a competitive inhibitor of dihydro-orotate synthesis though a variety of other pyrimidines had no effect on enzyme activity [99]. 

Dihydro-orotate-oxidizing activity in rat liver homogenates can be recovered completely in the mitochondrial fraction [103,104].With the exception of this system all the other enzymes of the orotate pathway appear to be present in the soluble cytosolic fraction. Dihydro-orotate dehydrogenase from rat liver was found to be located on the outer surface of the inner membrane of mitochondria [105]. Dihydro-orotate can diffuse freely from the cytosol into the mitochondria and orotate can diffuse freely from the mitochondria into the cytosol. Therefore no active transport of either dihydro-orotate or orotate is required in pyrimidine synthesis [105]. In addition to inhibiting dihydro-orotase, orotic acid strongly blocks [103] dihydro-orotate oxidation. 

Dihydro-1,3-diazine-2,4-dione-6-carboxylic acid (orotic acid)... 

Five of the enzymes of UMP biosynthesis exist in the soluble fraction of Ehrlich ascites carcinoma as two enzyme complexes [143]. One complex contains the first three enzymes of the pathway, carbamoyl phosphate synthetase, aspartate carbamoyltransferase and dihydro-orotase and has an apparent molecular weight of 800000 to 850000 daltons. The second enzyme complex contains orotate phosphoribosyltransferase and orotidylic acid decarboxylase and sediments in a sucrose gradient with 30% dimethyl sulphoxide and 5% glycerol with an apparent molecular weight of 105 000 to 115000 daltons [143]. 

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