A study on pyrimidine biosynthesis

Birnie, George David (1960) A study on pyrimidine biosynthesis. PhD thesis, University of Glasgow.

Abstract

The pathways of biosynthesis of the pyrimidine nucleotides uridino-5,-phosphate and cyt1dine-51-phosphate have been elucidated in recent years. The object of the present study was the development of a cell-free system in which the synthesis of thymine, thymidine or thymidylic acid could be investigated, with particular reference to the mechanism whereby the pyrimidine ring undergoes methylation. The organisms used were Escheriddhia soli PA/150 Esch. coli 113/3 Esch. coli 15T- and Bacillus subtilis N.O.I.B. 8059. The ability of Esch. coli PA/15 to catalyse the synthesis of mothionine from homocysteine and sorine, a reaction formally analogous to the methylation o+/- uracil or doonyuridine, was confirmed. Attempts were made to synthesise thymine or thymidine from uracli or deoxyuridino using Esch. coli PA/15 and B. subtilis 8059 under the same conditions as those involved in mebhionino synthesis. No evidence was obtained for the synthesis of thymine or thymidine in this system using (1) a micro-biological assay system employing Esch. Coli.15T- as assay organism or (ii) extensive paper chromatographic analysis, with the use of 3-14C-serine as the one-carbon unit precursor. The use of the cofactors folic acid, N10-Extensive examination or these systems for inter-mediates on the biosynthetic pathway to thymidyllo acid was carried out, but no intermediates of the 5-hydroxy-methyl-pyrimidine type were identified. Experiments using extracts of E. coil 15T-, a thymine- or thymidine-less mutant, as the enzyme source wore carried out but again no bhymidylic acid precursors of the 5-hydroxymethyl-pyrimidine type were identified. Successful chemical syntheses of 5- hydroxymethyluracil and 5-hydroxymethyldeoxyuridine wore achieved and the structure of the latter was confirmed by degradation studies. Attempts to synthesis 5-hydroxymethyldeoxy-uridylic acid were unsuccessful. Evidence was obtained that thymidine-W-triphosphate was formed in systems in which thymidylic acid was synthesised, but no in vitro synthesis of DNA-thymine was, detected. In the same systems. A comparison of serine, formaldehyde and formate as one-carbon unit precursors revealed that serine was a much more prolific source of one-carbon units than either formaldehyde or formate. A similar comparison of deoxyuridine, uridine and deoxyuridylic acid as ono carbon unit acceptors showed that uridino was a less efficient acceptor than either deoxyuridine or deoxy-uridylic acid but no significant difference between deoxyuridine and deoxyuridylic acid was detected. The effect of Vitamin B12 on thymidylic acid biosynthesis was also investigated using Esch. Coli 113/3, a methionine- or vitamin B12-less mutant, but not unequivocal evidence for a vitamin B12 effect was obtained with extracts of Esch. Coli 113/3 cells which had been depleted of vitamin B12 by serial sub-culturing in a methionine medium. Further extensive investigation of the 14C-thymidylic acid synthesized in these systems revealed that a large proportion of the 14C-thymidylic acid isolated was not, in fact, authentic thymidylic acid. Attempts to elucidate the structure of the contaminant have been carried out. Degradative and autoradiographic studies have led to t tentative identification of B-amino-iso-butryic acid and B-ureido-iso-butyric acid as hydrolytic products of the thymidylic acid contaminant. On this evidence, the contaminant appears to be the 4:5-dihydro-derivative of thymidylic acid. Parallel investigation of the degradation of thymine and thymidylic acid by extracts of Esch. Coli PA/15 has provided spectrophotometric evidence that the organism catalyses the reduction of the pyrimidine ring across the 4:5-double bond.Thymidine does not appear to be a substrate for this reduction system.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: G W Crosbie
• Keywords: Biochemistry
• Date of Award: 1960
• Depositing User: Enlighten Team
• Unique ID: glathesis:1960-73143
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73143