Incorporation of 4-amino-5-hydroxymethylpyrimidine into thiamine by microorganisms

One possible route for the biosynthesis of the (4-amino-2-methyl-5-pyrimidyl)-methyl moiety of thiamine would involve the formation of a methyl group on the demethylated pyrimidine, 4-amino-5-hydroxymethylpyrimidine, before its incorporation into thiamine. This possibility was tested by preparing the 4-amino-5-hydroxymethylpyrimidine and feeding it to Escherichia coli. Bacillus subtilis, and Saccharomyces cerevisiae. Analysis of the thiamine produced by these organisms showed that 4-amino-5-hydroxymethylpyrimidine was readily incorporated into thiamine without the addition of a methyl group, and no evidence was found for the conversion of this pyrimidine into normal methylated pyrimidine. Substitution of the demethylated thiamine for thiamine had no effect on the growth rate or the yield of E. coli cells. Complete substitution of the thiamine with the (4-amino-5-pyrimidyl)-methyl moiety was possible in an E. coli pur I mutant. The extent of incorporation of the demethylated pyrimidine decreased in some organisms and increased in others by the addition of adenine to the growth medium; this difference led to a simple test to separate organisms that use 5-aminoimidazole ribonucleotide for the biosynthesis of thiamine pyrimidine from those that do not.