Precursor of carbon atom five and hydroxymethyl carbon atom of the pyrimidine moiety of thiamin in Escherichia coli.

Abstract

The pyrimidine moiety of thiamin can be synthesized in bacteria from 5-aminoimidazole ribotide (AIR), an intermediate in purine biosynthesis. To transform the imidazole ring of AIR to the pyrimidine, the bond between C-4 and C-5 of the imidazole ring should be ruptured and a two-carbon unit should be inserted. We investigated a precursor of the two-carbon unit which should be inserted to form the pyrimidine. To examine the possibility of the two-carbon compound as a precursor which is formed from a three-carbon intermediate in the glycolytic pathway and its three-carbon derivative by decarboxylation in cells, we studied the incorporation of [2-14C]glycerol, [2-14C]pyruvate, [U-14C]alanine and [3-14C]serine into the pyrimidine by Escherichia coli. Glycerol, pyruvate and alanine were not incorporated into the pyrimidine. Radioactive carbon of [3-14C]serine was incorporated into C-2 of the pyrimidine via one-carbon units pool. Then, we investigated the possibility of a ribose moiety of AIR being a precursor of the two-carbon unit. As ribose has low permeability into E. coli cells, the ribose moiety of AIR was labeled with [14C]glucose which was added to the growth medium. The results show that two radioactive carbons of [U-14C]glucose were incorporated into the pyrimidine and radioactive carbon of [6-14C]glucose was incorporated into hydroxymethyl carbon attached to C-5 of the pyrimidine. The dilution rates of the specific radioactivity of the pyrimidine from [U-14C]glucose and [6-14C]glucose well coincide with those of the ribose moiety of the nucleotide (AMP). Radioactive carbon of [1-14C]glucose was not incorporated into the pyrimidine and nucleotide. It is concluded that the two-carbon fragment derived from C-4' and C-5' of the ribose moiety of AIR can be incorporated into the pyrimidine moiety of thiamin as the precursor of C-5 and hydroxymethyl carbon.