BIOSYNTHESIS OF THE PYRIMIDINE MOIETY OF THIAMIN IN ESCHERICHIA-COLI - INCORPORATION OF STABLE ISOTOPE-LABELED GLYCINES

Methods are described for the cleavage, extraction and subsequent gas chromatographic-mass spectrometric analysis of the pyrimidine moiety of thiamin as 2-methyl-4-amino-5-[(ethylthio)methyl]pyrimidine. The methods are of a general nature and can be applied to any system. Using these methods to evaluate the incorporation of 13C-, 15N- and 2H-labeled glycines into the pyrimidine moiety of thiamin by Escherichia coli, we established that the nitrogen and carbon 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 has established that the glycine nitrogen 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 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 thiamin in E. coli. © 1979, American Chemical Society. All rights reserved.