Orotate transferases

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [168], as well as an increase in rate of turnover of these polynucleotide structures [169,170]. Further experiments in chicks indicate that orotic acid, vitamin B12 and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [171]. Both orotic acid and vitamin Bu increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [172]. The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B12 [173]. In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [174—176]. It is therefore postulated that there may be a common role of orotic acid and vitamin Bj2 at the level of the transcription process in m-RNA biosynthesis [174—176]. 

Recently, Isshi et al. demonstrated that high levels of orotate phosphorybosyl transferase (OPRT) may be associated with increased sensitivity to 5-FU based chemotherapy (79). OPRT catalyzes the reduction of FUDP to the actively TS-inhibiting metabolite FdUMP, indicating a role for chemosensitivity to 5-FU. A recent study by Ichikawa et al. indicated that a newly identified SNP of OPRT exon 3 (G-to-A substitution) may be critical to predict toxicity to 5-FU based chemotherapy (80). 

Isshi K, Sakuyama T, Gen T et al. Predicting 5-FU sensitivity using human colorectal cancer specimens comparison of tumor dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase activities with in vitro chemosensitivity to 5-FU. Int J Clin Oncol 2002 7 335-342. 

Fig. lA. Anabolic and catabolic pathways of 5-FU. DPD dihydropyrimidine dehydrogenase, DP di-hydropyrimidinase, pUP beta-ureidopropionase, UP uridine phosphorylase, OPRT orotate phospho-ribosyl transferase, UK uridine kinase, TP thymidine phosphorylase, TK thymidine kinase, RNR ribonucleotide reductase. The three active metabolites (shown in rectangles) are FdUMP (5-fluoro-2 -deoxyuridine 5 -monophosphate) inhibiting TS (thymidylate synthase), and FUTP (5-fluorouridine 5 -triphosphate) and FdUTP (5-fluoro 2 -deoxyuridine 5 -triphosphate) interfering with RNA and DNA, respectively. 

Orotate phosphoribosyl transferase and OMP decarboxylase are separate domains of a single polypeptide—UMP synthase. 

In hereditary orotic aciduria, orotic acid is excreted in the urine because the enzymes that convert it to uridine monophosphate, orotate phosphoribosyl transferase and orotidine 5 -phosphate decarboxylase, are defective (see Figure 7-20). Pyrimidines cannot be synthesized, and therefore, normal growth does not occur. Oral administration of uridine bypasses the metabolic block and provides the body with a source of pyrimidines. 

Reaction of aspartic acid (14) with carbamoyl phosphoric acid (17) in the presence of the allosteric enzyme aspartate carbamoyltransferase (aspartate transcar-bamoylase) gives N-carbamoyl aspartic acid (18), which is cyclised to L-dihy-droorotic acid (19) by dihydroorotase. Oxidation of L-dihydroorotic acid by flavoprotein, orotate reductase gives orotic acid (20), which reacts with 5-phosphori-bosy 1-1-pyrophosphate (PRPP) in the presence of orotate phosphoribosyl transferase to form orotidine 5 -monophosphate (OMP, 21). Decarboxylation of OMP by orotid-ine 5 -phosphate decarboxylase yields uridine 5 -monophosphate (UMP, 22), which acts as precursor for the cytidine nucleotides (CTP) (Chart 6). 

Although the exact nature of the various enzymes involved in pyrimidine biosynthesis is not fully worked out, it seems that leishmania and trypanosomes possess phosphoribosyl transferase, which is specific for uracil. This makes the protozoal phosphoribosyl transferase distinct from the mammalian orotate phosphoribosyl transferase and, therefore, may be explored in protozoal chemotherapy. 

Fluorouracil (5-FU) requires enzymatic conversion to the nucleotide (ribosylation and phosphorylation) in order to exert its cytotoxic activity. Several routes are available for the formation of floxuridine monophosphate (FUMP). 5-FU may be converted to fluorouridine by uridine phos-phorylase and then to FUMP by uridine kinase, or it may react directly with 5-phosphoribosyl-l-pyrophosphate (PRPP), in a reaction catalyzed by orotate phosphoribosyl transferase, to form FUMP. Many metabolic pathways are available to FUMP. As the triphosphate FUTP, it may be incorporated into RNA. An alternative reaction sequence... 

Fig. 6. Diagram from Scapin el al. (Biochemistry 34 (1995) 10744-10754) illustrating the location of bound orotic acid (light atoms), of orotic acid glycosidically linked in orotidyl 5 -phospho-/8-D-riboside (dark atoms), and of pyrophosphate (light atoms) oriented to form the a-5 -phosphoriboside in oro-tate 5 -phosphoribosyl transferase.

Fig. 7. Electrostatic potential surface of the active-site region of orotate 5 -phosphoribo-syl transferase complexed with orotidyl /3-5 -phosphori bosidc. Reproduced from Scapin el al., (1995) Biochemistry 34,10744-10754, with permission of the American Chemical Society.

FIG. 6.12 Pyrimidine de novo synthesis pathway. Enzymes are as follows (1) carbamoyl-phosphate synthetase II (2) asparate carbamoyl-transferase (3) dihydro-orotase (4) dihydro-orotate oxidase (5) orotate phosphoribosyltransferase (6) orotidine-5 -phosphate decarboxylase (7) nucleoside monophosphate kinase (8) nucleotide diphospho kinase (9) CTP synthetase. 

In the next step of pyrimidine biosynthesis, the entire aspartate molecule adds to carbamoyl phosphate in a reaction catalyzed by aspartate transcarbamoylase. The molecule subsequently closes to produce a ring (catalyzed by dihydroorotase), which is oxidized to form orotic acid (or its anion, orotate) through the actions of dihydroorotate dehydrogenase. The enzyme orotate phosphoribosyl transferase catalyzes the transfer of ribose 5-phosphate from PRPP to orotate, producing orotidine 5 -phosphate, which is decarboxylated by orotidylic acid dehydrogenase to form... 

Orotate condenses with PRPP in a reaction catalyzed by orotate phosphoribosyl transferase to form the nucleotide orotidylate (OMP). Orotidylate decarboxylase converts OMP to the more abundant nucleotide UMP. The reaction occurs during de novo pyrimidine biosynthesis and is therefore not a salvage reaction. 

URAS Orotate phosphoribosyl transferase 5-FOA selection for Ura" strains [77]... 

The first reaction is catalysed by orotate phosphoribosyltransferase (orotidine 5 -phosphate pyrophosphate phosphoribosyltransferase, EC 2.4.2.10) which is readily reversible. The equilibrium constant for the forward reaction [109] is about 0.1. The reaction is specific for orotate (the enzyme usually does not accept uracil) and some synthetic analogues of orotic acid (Chapter 6). Orotate phosphoribosyltransferase activity was found in many animal tissues [110] and there are several phosphoribosyl-transferases of broad specifity which are distinct from the enzyme involved in the orotate pathway [111-113]. 

H34 Hoffman, D. H. and Sweeney, M. J. Orotate phosphor-ibosyl transferase and orotidylic acid decarboxylase activities in liver and Morris hepatomas. Cancer Res., 33, 1109-1112 (1973)... 

Ml Macleod, P., Mackenzie, S. and Scriver, C. R. Partial ornithine carbamyl transferase deficiency, an inborn error of the urea cycle presenting as orotic aciduria in a male infant. Can. Med. Assoc. J., 107, 405-408 (1972)... 

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