The Enzymatic Conversion of Uracil 5-Carboxylic Acid to Uracil and Carbon Dioxide

Insights into the Biochemistry, Evolution, and Biotechnological Applications of the Ten-Eleven Translocation (TET) Enzymes

“…A more definitive role for TETs in active demethylation was established two years later, when two groups independently demonstrated, using a variety of analytical techniques, that the three mouse TET (mTET) paralogues could catalyze further oxidation of 5hmC in oligonucleotides and gDNA to produce 5fC and 5caC in an Fe 2+ - and aKG-dependent manner. , The results of these studies rationalized the observation of 5fC in mouse embryonic stem cell (mESC) gDNA in an earlier report by Pfaffeneder et al The discovery that TETs could produce 5caC triggered relatively fruitless searches for a decarboxylase similar to the thymine salvage enzyme isoorotate decarboxylase − that would catalyze decarboxylation of 5caC and re-establish C on genomic sites without resorting to the resource intensive TDG-BER pathway. One study found that treatment of [1,3- 15 N]­5caC-labeled DNA with mESC extract resulted in product containing isotopically labeled C, suggesting that such a decarboxylase may exist .…”

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

“…The 2'-hydroxylase was typically purified as shown in Table I. While the calcium phosphate gel fractionation procedure (steps 2 and 3) freed the 2'-hydroxylase from uracil-5-carboxylic acid decarboxylase (Palmatier et al, 1970) no detectable separation of any of the other enzymes, involved in the oxidative demethylation of thymidine, was achieved until the DEAE-cellulose chromatography step was carried out. Three peaks of enzymatic activity were eluted from the DEAE-cellulose column as depicted in Figure 1.…”

“…Similarly, 5-hydroxymethyluracil appears to inhibit the conversion of 5-formyluracil to uracil-5-carboxylic acid (Holme etal., 1971). The uracil-5-carboxylic acid produced, when 5-formyluracil was used as substrate, was not converted to uracil since the decarboxylase had been removed during the purification procedure (Palmatier et al, 1970).…”

“…11, no. 11, 1972 tion of its radioactivity with the use of a scintillation counter have also been described (Palmatier et al, 1970). To further establish the identity of the enzymatic product of a-[5-14C]ketoglutarate in the 2'-hydroxylase reaction, a standard incubation mixture was prepared using nonlabeled thymidine and the 2 '-hydroxylase fraction which had been concentrated with ammonium sulfate using albumin as a "carrier," as indicated above.…”

“…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).…”