Supplementary Material for: Polyphosphate Kinases Phosphorylate Thiamine Phosphates

Polyphosphate kinases (PPKs) catalyze the reversible transfer of the γ-phosphate moiety of ATP (or of another nucleoside triphosphate) to a growing chain of polyphosphate (polyP). In this study we describe that PPKs of various sources are additionally able to phosphorylate thiamine diphosphate (ThP2) to produce thiamine triphosphate (ThP3) and even thiamine tetraphosphate (ThP4) in vitro. Furthermore, all tested PPK2s, but not PPK1s, were able to phosphorylate thiamine monophosphate (ThP1) to ThP2 and ThP3 although at low efficiency. The predicted masses and identities of the mono- and oligo-phosphorylated thiamine metabolites were identified by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Moreover, the biological activity of ThP2, that was synthesized by phosphorylation of ThP1 with polyP and PPK, as a cofactor of ThP2-dependent enzymes (here transketolase TktA from Escherichia coli) was confirmed in a coupled enzyme assay. In conclusion, our study shows that PPKs are promiscuous enzymes that are presumably involved in the formation of a variety of phosphorylated metabolites in vivo.