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 greater than 60% but deuterium from [2-(2)H2]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.
D002247:Carbon Isotopes; D002849:Chromatography, Gas; D003903:Deuterium; D004926:Escherichia coli / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005998:Glycine / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes; D011743:Pyrimidines / Q000096:biosynthesis*; D013831:Thiamine / Q000096:biosynthesis*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011743:Pyrimidines; D003903:Deuterium; D005998:Glycine; D013831:Thiamine