Orotate phosphoribosyltransferase

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

Orotate phosphoribosyltransferase (reaction 5, Figure 34-7) converts the drug allopurinol (Figure 33-12) to... 

The orotic aciduria that accompanies Reye s syndrome probably is a consequence of the inabifity of severely damaged mitochondria to utifize carbamoyl phosphate, which then becomes available for cytosofic overproduction of orotic acid. Type I orotic aciduria reflects a deficiency of both orotate phosphoribosyltransferase and orotidylate decarboxylase (reactions 5 and 6, Figure 34—7) the rarer type II orotic aciduria is due to a deficiency only of orotidylate decarboxylase (reaction 6, Figure 34-7). 

Orotate phosphoribosyltransferase (OPRT) catalyses the conversion of 5-FU to fluorouridine monophosphate (FUMP), which is subsequently phosphorylated to activated fluorouridine triphosphate (FUTP). FUTP incorporates into RNA and thereby compromises RNA processing and function (Fig. 1). 

Low activities of orotidine phosphate decarboxylase and orotate phosphoribosyltransferase result in abnormal growth, megaloblastic anemia and the excretion of large amounts of orotate in the urine. 

The last two steps in the biosynthesis of the mononu- cl cotide uridine 5 -monophosphate (UMP) are catalyzed by (1) orotate phosphoribosyltransferase (OPRTase) and (2) orotate 5 -monophosphate decarboxylase (OMPDase). 

OMPDase = orotate 5 -monophosphate decarboxylase OPRTase = orotate phosphoribosyltransferase. b Units refer to total amount of enzyme activity. 

In the third step, the pyrimidine ring is closed by dihy-droorotase to form 1-dihydroorotate. Dihydroorotate is then oxidized to orotate by dihydroorotate dehydrogenase. This flavoprotein in some organisms contains FMN and in others both FMN and FAD. It also contains nonheme iron and sulfur. In eukaryotes it is a lipoprotein associated with the inner membrane of the mitochondria. In the final two steps of the pathway, orotate phosphoribosyltransferase yields orotidine-5 -phosphate (OMP), and a specific decarboxylase then produces UMP. 

Low activities of orotidine phosphate decarboxylase and (usually) orotate phosphoribosyltransferase are associated with a genetic disease in children that is characterized by abnormal growth, megaloblastic anemia, and the excretion of large amounts of orotate. When affected children are fed a pyrimidine nucleoside, usually uridine, the anemia decreases and the excretion of orotate diminishes. A likely explanation for the improvement is that the ingested uridine is phosphorylated to UMP, which is then converted to other pyrimidine nucleotides so that nucleic acid and protein synthesis can resume. In addition, the increased intracellular concentrations of pyrimidine nucleotides inhibit carbamoyl phosphate synthase, the first enzyme in the. naibwav of aro-tate synthesis. 

In eukaryotes, carbamoyl phosphate synthase is inhibited by pyrimidine nucleotides and stimulated by purine nucleotides it appears to be the most important site of feedback inhibition of pyrimidine nucleotide biosynthesis in mammalian tissues. It has been suggested that under some conditions, orotate phosphoribosyltransferase may be a regulatory site as well. 

The first two steps in the biosynthesis of tryptophan in Salmonella typhimurium involve the enzyme complex anthranilate synthase-phosphoribosyltransferase, which is a tetramer having two subunits of each enzyme. The anthranilate synthase catalyzes reaction (7) and the phos-phoribosyltransferase catalyzes two reactions the N-terminal portion cleaves glutamine to glutamate giving NH3 for the anthranilate synthase, while the C-terminal portion catalyzes reaction (8).3,1,312 All these reactions require M2+ cations. Orotate phosphoribosyltransferase binds four Mn2+ ions in a cooperative fashion kinetic data have been interpreted in a scheme where both metal-free and metal-containing enzyme catalyze the reaction.313... 

Such conversions in mammals are not very efficient, however, with the exception of, perhaps, the orotate phosphoribosyltransferase, which is a component of the pyrimidine nucleotide biosynthetic pathway (Figure 10.9). A very active uracil phosphoribosyltransferase has been isolated from microorganisms. It converts uracil to UMP using PRPP. 

There are two multifunctional proteins in the pathway for de novo biosynthesis of pyrimidine nucleotides. A trifunctional protein, called dihydroorotate synthetase (or CAD, where the letters are the initials of the three enzymatic activities), catalyzes reactions 1, 2 and 3 of the pathway (HCC>5"- CAP— CA-asp—> DHO Fig. 15-15). The enzymatic activities of carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase, are contained in discrete globular domains of a single polypeptide chain of 243 kDa, where they are covalently connected by segments of polypeptide chain whch are susceptible to digestion by proteases such as trypsin. A bifunctional enzyme, UMP synthase, catalyzes reactions 5 and 6 of the pyrimidine pathway (orotate— OMP—> UMP Fig. 15-15). Two enzymatic activities, those of orotate phosphoribosyltransferase and OMP decarboxylase, are contained in a single protein of 51.5 kDa which associates as a dimer. 

Fig. 15-15 The de novo pyrimidine biosynthetic pathway. CAP, carbamoyl phosphate CA-asp, /V-carbamoyl-L-aspartate DHO, L-dihydroorotate Oro, orotate OMP, orotidine 5 -monophosphate. Enzymes (1) carbamoyl phosphate synthetase II (2) aspartate transcarbamoylase (3) dihydroorotase, (4) dihydroorotate dehydrogenase (5) orotate phosphoribosyltransferase (6) OMP decarboxylase (7) nucleoside monophosphate kinase (8) nucleoside diphosphate kinase (9) CTP synthetase.

A genetic deficiency of the orotate phosphoribosyltransferase results in accumulation of orotate and its excretion in the urine. 

The metabolic interrelationship between mitochondrial carbamoyl phosphate synthesis to urea formation and to cytosolic carbamoyl phosphate channeled into pyrimidine biosynthesis. In ornithine transcarbamoyiase (OTC) deficiency, mitochondrial carbamoyl phosphate diffuses into the cytosol and stimulates pyrimidine biosynthesis, leading to orotidinuria. Administration of allopurinol augments orotidinuria by increasing the flux in the pyrimidine biosynthetic pathway. CPS = Carbamoyl phosphate synthase, AT = aspartate transcarbamoyiase, D = dihydroorotase, DH = dihydroorotate dehydrogenase, OPRT = orotate phosphoribosyltransferase, XO = xanthine oxida.se,... 

Figure 27-27), aspartate transcarbamoylase, and dihydroorotase activity. Each subunit of Pyr 1-3 has a molecular weight of 200,000-220,000, and the native enzyme exists as multiples of three subunits. The second gene codes for dihydroorotate dehydrogenase which is located on the outer side of the inner mitochondrial membrane. Dihydroorotate, the product of Pyr 1-3, passes freely through the outer mitochondrial membrane and converted to orotate. Orotate readily diffuses to the cytosol for conversion to UMP. The third gene codes for another multifunctional polypeptide known as UMP synthase (Pyr 5,6). Pyr 5,6 (M.W. 55,000) contains orotate phosphoribosyltransferase and orotidylate (orotidine-5 -monophosphate) decarboxylase activity. Use of multifunctional polypeptides is very efficient, since the intermediates neither accumulate nor become consumed in side reactions. They are... 

Schematic representation of the intracellular location of the six enzymes of UMP biosynthesis in animals. Pyr 1-3 = 1, Carbamoyl phosphate synthetase II 2, aspartate transcarbamoylase 3, dihydroorotase 4, dihydroorotate dehydrogenase Pyr 5,6 = 5, orotate phosphoribosyltransferase 6, orotidine-5 -monophosphate decarboxylase.

A 4-year-old girl presents in the clinic with megaloblastic anemia and failure to thrive. Blood chemistries reveal orotic aciduria. Enzyme measurements of white blood cells reveal a deficiency of the pyrimidine biosynthesis enzyme orotate phosphoribosyltransferase and abnormally high activity of the enzyme aspartate transcarbamoylase. Which one of the following treatments will reverse all symptoms if carried out chronically ... 

The answer is e. (Murray, pp 375-401. Scriver, pp 2663-2704. Sack, pp 121-138. Wilson, pp 287—320.) Orotic aciduria is the buildup of orotic acid due to a deficiency in one or both of the enzymes that convert it to UMP Either orotate phosphoribosyltransferase and orotidylate decarboxylase are both defective, or the decarboxylase alone is defective. UMP is the precursor of UTP, CTP, and TMP All of these end products normally act in some way to feedback-inhibit the initial reactions of pyrimidine synthesis. Specifically, the lack of CTP inhibition allows aspartate transcarbamoylase to remain highly active and ultimately results in a buildup of orotic acid and the resultant orotic aciduria. The lack of CTP, TMP, and UTP leads to a decreased erythrocyte formation and megaloblastic anemia. Uridine treatment is effective because uridine can easily be converted to UMP by omnipresent tissue kinases, thus allowing UTP, CTP, and TMP to be synthesized and feedback-inhibit further orotic acid production. 

Krungkrai, S. R., Aoki, S., Palacpac, N. M., Sato, D., Mitamura, T., Krungkrai, J., and Horii, T. (2004). Human malaria parasite orotate phosphoribosyltransferase Functional expression, characterization of kinetic reaction mechanism and inhibition profile. Mol. Biochem. Parasitol. 134, 245-255. 

Krungkrai, S. R., DelFraino, B. J., Smiley, J. A., Prapunwattana, P., Mitamura, T., Horii, T., and Krungkrai, J. (2005). A novel enzyme complex of orotate phosphoribosyltransferase and orotidine 5 -monophosphate decarboxylase in human malaria parasite Plasmodium falciparum Physical association, kinetics, and inhibition characterization. Biochemistry 44, 1643-1652. 

The reaction of carbamoyl phosphate with aspartate to produce W-carbamo-ylaspartate is the committed step in pyrimidine biosynthesis. The compounds involved in reactions up to this point in the pathway can play other roles in metabolism after this point, A -carbamoylaspartate can be used only to produce pyrimidines—thus the term committed step. This reaction is catalyzed by aspartate transcarbamoylase, which we discussed in detail in Ghapter 7 as a prime example of an allosteric enzyme subject to feedback regulation. The next step, the conversion of A-carbamoylaspartate to dihydroorotate, takes place in a reaction that involves an intramolecular dehydration (loss of water) as well as cyclization. This reaction is catalyzed by dihydroorotase. Dihydroorotate is converted to orotate by dihydroorotate dehydrogenase, with the concomitant conversion of NAD to NADH. A pyrimidine nucleotide is now formed by the reaction of orotate with PRPP to give orotidine-5 -monophosphate (OMP), which is a reaction similar to the one that takes place in purine salvage (Section 23.8). Orotate phosphoribosyltransferase catalyzes this reaction. Finally, orotidine-5 -phosphate decarboxylase catalyzes the conversion of OMP to UMP... 

A bifunctional enzyme, UMP synthase, catalyzes reactions 5 and 6 of the pyrimidine pathway (orotate —> OMP — UMP Fig. 14-7). Two enzymic activities, those of orotate phosphoribosyltransferase and OMP decarboxylase, are contained in a single protein of 51.5 kDa that associates as a dimer. 

The disease results from an inherited deficiency of orotate phosphoribosyltransferase that causes the accumulation of orotate/orotic acid and the excretion of the excess in the urine. 

Orotate phosphoribosyltransferase Orotidine 5 -phosphate Uridine 5 -phosphate 6-hydroxyuridine 5 -phosphate Triosephosphate isomerase Angstrom Turnover number Michaelis constant Catalytic efficiency... 

Orotidine 5 -phosphate decarboxylase (ODCase, E. C. 4.1.1.23) catalyzes the decarboxylation of orotidine 5 -phosphate (OMP) to form uridine 5 -phos-phate in the sixth and final step of pyrimidine biosynthesis (Fig. 1) [1]. The discovery of ODCase in 1954 followed the identification, three years earlier, of orotic acid as the metabolic precursor of nucleic acids [2, 3]. ODCase is a distinct, monofunctional polypeptide in bacteria and fungi, whereas in mammals it combines with orotate phosphoribosyltransferase (OPRTase) to form the bifunctional enzyme UMP synthase. Human deficiencies in either OPRTase or ODCase activity result in an autosomal recessive disorder called hereditary orotic aciduria [4]. The disease is characterized by depleted levels of pyrimidine nucleotides in the blood and by the appearance of crystalline... 

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