Biosynthesis of the pyrimidine moiety of thiamin in Escherichia coli: incorporation of stable isotope-labeled glycines

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Abstract
Methods are described for the cleavage, extraction and subsequent gas chromatographic-mass spectrometric analysis of the pyrimidine moiety of thiamine as 2-methyl-4-amino-5-[(ethylthio)methyl]pyrimidine. The methods are of a general nature and can be applied to an system. Using these methods to evaluate the incorporation of 13C-, 15N- and 2H-labeled glycines into the pyrimidine moiety of thiamine by E. coli, the N and C atoms of glycine are incorporated as a unit into the pyrimidine. 13C- and 15N-labeled glycines are incorporated at > 60% but deuterium from [2-2H2]glycine was incorporated at only 18%. A detailed analysis of the mass fragmentation pattern of the pyrimidine derivative established that the glycine N atom supplies the N-1 of the pyrimidine and that the C-1 and C-2 of the glycine supplies the C-4 and C-6 of the the pyrimidine, respectively. This evidence is consistent with the substitution of a C2 unit between the C-5 and C-4 of the 4-aminoimidazole ribonucleotide precursor during the biosynthesis of the pyrimidine moiety of thiamine in E. coli.

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