Several Polyphosphate Kinase 2 Enzymes Catalyse the Production of Adenosine 5′‐Polyphosphates

Abstract

Polyphosphate kinases (PPKs) are involved in many metabolic processes; enzymes of the second family (PPK2) are responsible for nucleotide synthesis fuelled by the consumption of inorganic polyphosphate. They catalyse the phosphorylation of nucleotides with various numbers of phosphate residues, such as monophosphates or diphosphates. Hence, these enzymes are promising candidates for cofactor regeneration systems. Besides adenosine 5′‐triphosphate, PPK2s also catalyse the synthesis of highly phosphorylated nucleotides in vitro, as shown here for adenosine 5′‐tetraphosphate and adenosine 5′‐pentaphosphate. These unusually phosphorylated adenosine 5′‐polyphosphates add up to 50 % of the whole adenosine nucleotides in the assay. The two new products were chemically synthesised to serve as standards and compared with the two enzymatically produced compounds by high‐performance ion chromatography and 31P NMR analysis. This study shows that PPK2s are highly suitable for biocatalytic synthesis of different phosphorylated nucleotides.

Class III Polyphosphate Kinase 2 Enzymes Catalyze the Pyrophosphorylation of Adenosine‐5′‐Monophosphate

“…In a recent study, four PPK2 enzymes derived from classes I, II, and III were shown to produce adenosine 5′‐polyphosphate molecules, such as adenosine tetra‐ (AP4) and pentaphosphate (AP5) by using AMP or ADP and polyphosphate as substrates . We also observed a similar result with DtCs.…”

“…In addition, DtCs showed similar substrate preference to that of another previously characterized enzyme belonging to the same cluster. Our results provide a further understanding of the activity of the PPK2 class III enzymes, and provide clues to the underlying mechanism of the synthesis of adenosine polyphosphate observed in a recent study …”

“…In another recent study, adenosine polyphosphate was shown to be produced by several PPK2 enzymes. Interestingly, this was the case for all classes of PPK2 enzymes.…”

“…Based on the proposed mechanism of PPK2 class III enzymes, we assumed that AMP was first phosphorylated to ADP, followed by an additional phosphorylation to generate ATP. Because more ATP was generated than ADP, we hypothesized that not only AMP, but also ADP, was a favorable substrate of DtCs.…”

Enzymatic One‐Step Reduction of Carboxylates to Aldehydes with Cell‐Free Regeneration of ATP and NADPH

“…PPKs recently reported to be also able to transfer more than one phosphate unit in a single event were not considered for use in this work. [20] In addition to the selection of PPKs, shorter polyphosphate chains are expected to be no efficient donors [15] and therefore, an appropriate excess of polyP mustbep rovided. Figure 2c learly shows that am inimum amount of 40 mm polyP (calculation based on ortho-phosphate units) is required to reach complete reduction of 10 mm of 1a in vitro.…”