The enzyme catalyses the penultimate reaction in thiamine de novo biosynthesis, condensing the pyrimidine and thiazole components. The enzyme is thought to accept the product of EC 188.8.131.52
, thiazole synthase, as its substrate. However, it has been shown that in some bacteria, such as Bacillus subtilis, an additional enzyme, thiazole tautomerase (EC 184.108.40.206
) converts that compound into its tautomer 2-(2-carboxy-4-methylthiazol-5-yl)ethyl phosphate, and that it is the latter that serves as the substrate for the synthase. In addition to this activity, the enzyme participates in a salvage pathway, acting on 4-methyl-5-(2-phosphooxyethyl)thiazole, which is produced from thiamine degradation products. In yeast this activity is found in a bifunctional enzyme (THI6) and in the plant Arabidopsis thaliana the activity is part of a trifunctional enzyme (TH1).