Polyphosphate Kinases Phosphorylate Thiamine Phosphates

Abstract

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.

One-pot chemo-enzymatic synthesis and one-step recovery of length-variable long-chain polyphosphates from microalgal biomass

“…29 Recent phylogenetic analysis has identified three subtypes of PPK2s (class I, II, and III); 30,31 class I and II PPK2s catalyze the polyPdriven phosphorylation of either NDP or NMP, respectively, while class III PPK2s can phosphorylate both NDP and NMP, enabling direct NTP production from NMP. 32 Class I and II PPK2s have been used for in vitro biosynthesis of acetone, 33 aldehyde, 34 and thiamine phosphates 35 as well as biocatalytic regeneration of S-adenosyl-L-methionine, 36 while class III PPK2s have been used for cell-free protein synthesis and in vitro biocatalytic reactions that simultaneously require regeneration of both ATP and GTP. 37 In the PPK2-mediated A(G)TP regeneration system, long-chain polyP (100-mer), as opposed to short-chain polyP at the same molar content of total orthophosphate, can significantly enhance the cell-free protein yield.…”

One-pot chemo-enzymatic synthesis and one-step recovery of homogeneous long-chain polyphosphates from microalgal biomass

“…Recent phylogenetic analysis has identified three subtypes of PPK2s (class I, II, and III) 30,31 ; class I and II PPK2s catalyze the polyP-driven phosphorylation of either NDP or NMP, respectively, while class III PPK2s can phosphorylate both NDP and NMP, enabling direct NTP production from NMP 32 . Given their ability to generate NTPs from NDPs and NMPs, Class I and II PPK2s have been used for in vitro biosynthesis of acetone 33 , aldehyde 34 , thiamine phosphates 35 , and biocatalytic regeneration of S-adenosyl-L-methionine (SAM) using polyP as phosphate donor 36 .…”