Pathways of pyrimidine nucleotide biosynthesis in gravid Angiostrongylus cantonensis.

The incorporation of radioactive precursors into pyrimidine nucleotides via de novo and salvage pathways was measured in gravid Angiostrongylus cantonensis by HPLC and thin-layer chromatography. 14C-labelled orotate, uridine, uracil and deoxyuridine were traced to UMP, UDP, UTP, UDP-glucose, dTMP, CMP, CDP and CTP. 3H-labelled cytidine was also incorporated into both uracil and cytosine nucleotides in a ratio of 2:1. Cytosine was a major end-product for all the precursors. Cytosine nucleotides were probably formed from UTP by the action of CTP synthetase whose activity in crude cell-free extract was 31.5 +/- 4.9 pmol min-1 (mg protein)-1. It was dependent on glutamine, ATP and GTP and was inhibited by CTP. The total amount of pyrimidine nucleotides formed from uridine was 3 times of that from uracil. The presence of uracil in the metabolism of uridine indicates that UMP is formed by uracil phosphoribosyltransferase as well as by uridine kinase. UMP is a key intermediate for cytidylate and thymidylate biosynthesis in the gravid worms.

Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongylus cantonensis (Nematoda: Metastrongyloidea).

Gravid Angiostrongylus cantonensis can utilize radiolabelled bicarbonate, orotate, uracil, uridine and cytidine but not cytosine, thymine and thymidine for the synthesis of RNA and DNA. In cell-free extracts of the worm, a phosphoribosyltransferase was shown to convert orotate to OMP and uracil to UMP. A similar reaction was not observed with cytosine and thymine. Uridine was readily phosphorylated by a kinase but a similar reaction for thymidine and deoxyuridine was not found. Cytidine could be phosphorylated by a kinase or be deaminated by a deaminase to uridine. No deaminase for cytosine was detected. There was also no phosphotransferase activity for pyrimidine nucleosides in the cytosolic or membrane fractions. Pyrimidine nucleosides were, in general, converted to the bases by a phosphorylase reaction but only uracil and thymine could form nucleosides in the reverse reaction. The activity of thymidylate synthetase was also measured. These results indicate that the nematode synthesizes pyrimidine nucleotides by de novo synthesis and by utilization of uridine and uracil and that cytosine and thymine nucleotides are formed mainly through UMP. The thymidylate synthetase reaction appears to be vital for the growth of the parasite.

Angiostrongylus cantonensis: characterization of thymidylate synthetase.

Thymidylate synthetase (TS) is the only enzyme that catalyzes the formation of thymidine nucleotides in Angiostrongylus cantonensis. A fraction enriched in TS was obtained from the gravid nematode by gel filtration and affinity chromatography using methotrexate-agarose. TS, which was well separated from dihydrofolate reductase, has a relative molecular mass of 66 kDa. By electrophoresis in sodium dodecyl sulphate gel, a major protein band corresponding to 31 kDa was observed. This band was shown to be TS by comparing the electrophoretic mobility with an enzyme preparation bound with [6-3H]5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). Therefore, the enzyme is composed of two identical or very similar subunits. Velocity studies and product inhibition patterns revealed that the TS reaction undergoes a sequential mechanism in which 2'-deoxyuridine 5'-monophosphate (dUMP) is the first substrate added to the active site and thymidine 5'-monophosphate is the last product released. The apparent Km values for dUMP and 5,10-methylenetetrahydrofolate are 10 and 185 microM, respectively. FdUMP and trimethoprim inhibited the parasite TS competitively with dUMP and the Ki values of 23.5 nM and 852 microM, respectively. Methotrexate was a noncompetitive inhibitor of TS. At 0.2 mM 5,10-methylenetetrafolate, 1 mM methotrexate inhibited the activity by 74%.