Substrate specificity of the hydroxylase reaction in which thymidine is converted to thymine ribonucleoside

Molecular basis for the substrate specificity and catalytic mechanism of thymine-7-hydroxylase in fungi

“…The thymidine salvage pathway, which was first discovered in Neurospora crassa , consists of a series of catalytic reactions ( 18 ). After the hydrolysis of thymine ribonucleoside to ribose and thymine ( 19 ), the consecutive oxidations of thymine to 5hmU, 5fU and 5caU are catalysed by thymine-7-hydroxylase (T7H) ( 20 – 23 ), and the subsequent decarboxylation of 5caU to U is catalysed by isoorotate decarboxylase (IDCase) ( 24 – 26 ). Like the TET proteins, T7H is also a Fe 2+ and α-KG dependent dioxygenase ( 23 ).…”

Stoichiometry of the pyrimidine deoxyribonucleoside 2'-hydroxylase reaction and of the conversions of 5-hydroxymethyluracil to 5-formyluracil and of the latter to uracil-5-carboxylic acid

“…Subjection of extracts of Neurospora to a purification scheme, which includes calcium phosphate gel, ammonium sulfate, Sephadex G-150, and DEAE-cellulose fractionation procedures, yielded several enzyme fractions. One contained the 2'-hydroxylase and none of the other T X. he conversion of thymidine to the pyrimidines of RNA (Fink and Fink, 1962) is thought to be effected by the following enzymatic reactions: thymidine to thymine ribonucleoside, pyrimidine deoxyribonucleoside 2 '-hydroxylase reaction (Shaffer et al, 1968); thymine ribonucleoside to thymine plus ribose (Shaffer et al, 1972); thymine to 5-hydroxymethyl- * To whom correspondence should be addressed.…”

“…uracil, thymine 7-hydroxylase reaction (Abbott et al, 1967); 5-hydroxymethyluracil to 5-formyluracil (Abbott et al, 1968); 5-formyluracil to uracil-5-carboxylic acid (Watanabe et al, 1970); and uracil-5-carboxylie acid to uracil and C02, uracil-5-carboxylic acid decarboxylase reaction (Palmatier et al, 1970). The 2'-hydroxylase, which catalyzes the first step in this pathway, also converts deoxyuridine to uridine and requires a-ketoglutarate, Fe2+, ascorbate (Shaffer et al, 1968), and 02 for activity (Shaffer et al, 1972). Although the role of -ketoglutarate had not been studied in this reaction, it has been shown that -ketoglutarate is decarboxylated in the thymine 7-hydroxylase reaction ( Croskey et al, 1971) and that, in this process, molecular oxygen is incorporated into the 5-hydroxymethyluracil and succinate which are produced (Holme et al, 1971).…”

“…Since the resultant, partially purified enzyme preparation does not metabolize the thymine ribonucleoside produced from thymidine, the assay for the 2'-hydroxylase is much facilitated, and stoichiometry studies have been carried out with this preparation. The separation of this enzyme from the thymine 7-hydroxylase is interesting since it was a tenable hypothesis that thymidine and thymine interact with the same binding site in the hydroxylation reaction, even though the 2 '-hydroxylase activity was less stable during purification (McCroskey et al, 1971) and neither 5-hydroxymethyldeoxyuridine nor 5-hydroxymethyl uridine was detected as a product (Shaffer et al, 1972). Whether thymine 7-hydroxylase catalyzes each of the sequential reactions converting thymine to uracil-5-carboxylíc acid is not known, although a pyrimidineless mutant of Neurospora has been developed which will grow in media supplemented with 5-formyluracil but not in media in which 5-hydroxymethyluracil is the only pyrimidine source (Williams and Mitchell, 1969).…”