Thymidine 2'-hydroxylation in Neurospora crassa.

An enzyme preparation catalyzing the 2’-hydroxylation of thymidine was prepared from mycelial extracts of Neurospora crassa strain STA 4 (FGSC 2628) by chromatography on hydroxylapatite and DEAE-Sephadex A-50. The enzyme had an isoelectric point of 4.6 and a molecular weight of 47,000 (determined by gel filtration). The reaction product was identical with thymine riboside on ion exchange and thin layer chromatography. Paper chromatography after reduction and hydrolysis showed that the pentose moiety in the product was ribose. Of the pyrimidine deoxyribonucleosides tested as substrates, only those with oxygen at carbon atoms 2 and 4 were active, i.e. deoxyuridine, thymidine, 5-hydroxymethyldeoxyuridine, S-bromodeoxyuridine, and 6-azathymidine. Thymidylate was attacked only slowly. Purine deoxyribonucleosides, deoxyribose, and 1-ethyluracil were not substrates. No evidence for control by nucleotides was obtained. The enzyme had an absolute requirement for ferrous ion and 2-ketoglutarate, and ascorbate and catalase were stimulatory. 2-Ketoglutarate was oxidatively decarboxylated during the reaction. Together with the previously demonstrated oxygen requirement (SHAFFER, P. M., MCCROSKEY, R. P., PALMATIER, R. D., MIDGETT, R. J., AND ABBOTT, M.T. (1968) Biochem. Biophys. Res. Commun. 33,806) the present findings establish that thymidine 2’-hydroxylase belongs to the group of oxygenases which require a 2-keto acid for oxygen reduction (HOLME, E., LINDSTEDT, G., LINDSTEDT, S., AND TOFFT, M. (1968) Fed. Eur. Biochem. SOC. L&t. 2, 29; LINDBLAD, B., LINDSTEDT. G, LINDSTEDT, S., AND TOFFT, M. (1969) J. Amer. Chem. Sot. 91, 4604).