Orotate incorporation into nucleic acid pyrimidines

When N -orotic acid was injected into rats, the label was incorporated into the p3nimidines of RNA (346) as well as DNA (347). C -Orotic acid vas utilized similarly for p uimidine biosynthesis in rat liver and spleen (348, 349) and in yeast (28). Since orotic acid and pyrimidine nucleorides were incorporated into nucleic acid p3nrimidines while free pyrimidine bases were essentially inert biosynthetically (37, 303), it appeared that a ribose or phosphorylated ribose derivative of orotic acid might be a key intermediate in the formation of pyrimidine nucleosides and nucleotides. 

Entamoeba histolytica. There are several lines of evidence that E. histolytica is capable of pyrimidine de novo biosynthesis. It will incorporate orotate into nucleic acids (82), it has aspartate transcarbamoylase (83), and it will grow in medium that has been depleted of pyrimidines (84). It apparently can also meet its pyrimidine requirements by salvage (85). Its salvage pathways are similar to those of T. vaginalis, with the exception of the ability to salvage thymine (Fig. 6.15). Ribonucleotide reductase and thymidylate synthase activities are apparently present, since this parasite will grow in a pyrimidine deficient medium (84). 

Compound (LVII) is very rapidly excreted into the kidney. It is metabolised to 5-(3 -hydroxy-4 -aminophenyl)cytosine and its O-glucuronide, as well as derivatives acetylated on both amino groups [343]. It is a competitive inhibitor of succinate dehydrogenase (K = 9-2 x 10 m K,- = 5 0 X IO m), and it has been considered that this is its mode of action [343]. One role of this enzyme involves aspartate synthesis, via fumarate. This pyrimidine enhances the incorporation of orotic acid into nucleic acid but is not itself incorporated. 

Finally, as mentioned above, with the advent of a technology for manipulation of nucleotides, the free uridine nucleotides were recognized as intermediates in the incorporation of orotate into the nucleic acid pyrimidines 2), and it became apparent that the first product of this pathway to possess a nucleic acid base was uridylate. 

As early as 1949, it was demonstrated that injected or " C-labeled orotic acid was readily incorporated into DNA and RNA of mammalian tissue, indicating that orotic acid is a precursor of nucleic acid pyrimidine. The next step in pyrimidine biosynthesis is the formation of the first nucleotide in the sequence. It involves the reaction between ribosyl pyrophosphate and orotic acid to yield 5 -orotidylic acid the reaction is catalyzed by orotidylic pyrophosphorylase. Thus, the first steps of pyrimidine biosynthesis differ from the early steps of purine biosynthesis in at least two ways. Orotic acid, instead of being synthesized atom by atom as is the case for the purine ring, is made from the condensation of rather large molecules, namely, carbamyl phosphate and aspartic acid. Furthermore, all the steps of purine biosynthesis occur at the level of the nucleotide, but the the pyrimidine ring is closed at the level of the base. 

By measuring bicarbonate- C incorporatic into orotic aci as well as the incorporation of orotic acid- -C or uridine- C into nucleic acids it was possible to study the first and second part of pyrimidine biosynthesis and the salvage pathway separately. 

Eimeria tenella. It appears that the sporulated oocysts of E. tenella are capable of de novo pyrimidine synthesis since sporozoites incorporate " C-labeled aspartate and orotate into pyrimidine nucleotides (103). Besides de novo synthesis, E. tenella is also capable of pyrimidine salvage. E. tenella will incorporate uracil, cytidine and uridine but not thymidine into its nucleic acids (104). This parasite is dependent on UMP for the synthesis of TMP and thymidylate synthase activity is present in extracts of E. tenella (105). As with both Plasmodium and the kinetoplastids, the thymidylate synthase of E. tenella exists as a bifunctional protein (91). 

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