Thymine catabolism in Nocardia corallina : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Abstract

The oxidation of thymine, 5-methylbarbituric acid, methylmalonate and succinate was studied in cells grown on thymine, uracil, 5-methylbarbituric acid, barbituric acid, methylmalonate and succinate. In agreement with the results of Batt and Woods (1961) it was shown that thymine-grown cells oxidise thymine to 5-methylbarbituric acid which is in turn rapidly metabolised. Uracil-grown cells were shown to oxidise thymine to 5-methylbarbituric acid which accumulates and is metabolised only after thymine is all used up. Methylmalonate and succinate were oxidised significantly only in cells grown on the same carbon source, probably reflecting a requirement for permease. Metabolism of 5-methylbarbituric acid by cell-free extracts (but not by boiled cell-free extracts) was demonstrated, but the products remained unidentified. The use of [¹⁴C] 5-methylbarbituric acid in experiments with cell-free extracts was complicated by the gradual auto-oxidation of 5-methylbarbituric acid before and after the incubation period. The stability of 5-methylbarbituric acid under various experimental conditions was examined. Chromatographic separation of 5-methylbarbituric acid from growth medium resulted in up to 42% yield of 5-methylbarbituric acid. On storage, it was shown that [¹⁴C] 5-methylbarbituric acid was converted to 5-hydroxy-5-methylbarbituric acid and two other products, the major one of which was probably methyltartronyl urea. In long term incubations (1.5 to 6 hr.) of uracil-grown cells with [methyl-¹⁴C] thymine, most of the radioactivity incorporated in the ethanol soluble extract was in glutamate. Labelled methylmalonate was also produced, but in very low levels (this confirms the report of Mountfort, 1971). The long term incubation period and the presence of impurities in [¹⁴C] thymine made interpretation of results difficult. The remainder of the work was devoted to short term incubations by thymine-grown. cells with high specific activity [¹⁴C] thymine. The incorporation of ¹⁴C into various compounds was followed by two-dimensional thin layer chromatography (in phenol : water and n-butanol : acetic acid : water solvents) and autoradiography; and co-chromatography of radioactive compounds in various solvents. Kinetic studies with [2-¹⁴C] thymine suggest the following labelling sequence of thymine breakdown products: Thymine ⤑ 5-methylbarbituric acid ⤑ urea ⤑ CO₂. At very early times, an additional, rapidly metabolised compound appeared, and it is suggested that this may be thymidine. By a combination of results obtained by incubating cells with [2-¹⁴C] and [methyl-¹⁴C] thymine it could be shown that no 5-hydroxymethyluracil, uracil, barbituric acid, dihydrothymine, or β-ureidoisobutyrate were formed. This suggests that neither the reductive pathway nor the oxidative pathway via uracil operates in thymine-adapted Nocardia corallina under the experimental conditions used here. Kinetic studies with [methyl-¹⁴C] thymine suggests the following scheme of labelling of intermediates: Thymine ⤑ 5-methylbarbituric acid ⤑ methylmalonyl CoA (activated methylmalonate) ⤑ succinate ⤑ aspartate and alanine and then glutamate. A large pool of aspartate and glutamate present in Nocardia corallina acts as a trap for ¹⁴C. Activated methylmalonate was identified by hydrolysis to methylmalonate and also by treatment with hydroxylamine to form cethylnalonyl hydroxamate. Some activated succinate was also present since hydroxylanine treatment led to the formation of a hydroxamate, which on acid hydrolysis formed succinate.