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

Abstract

Polyphosphate kinase 2 (PPK2) transfer phosphate from inorganic polyphosphate to nucleotides. According to their activity, PPK2 enzymes are classified into three groups. Among them, class III enzymes catalyze both the phosphorylation of nucleotide mono‐ to diphosphates and di‐ to triphosphates by using polyphosphate, which is a very inexpensive substrate. Therefore, class III enzymes are very attractive for use in biotechnological applications. Despite several studies on class III enzymes, a detailed mechanism of how phosphate is transferred from the polyphosphate to the nucleotide remains to be elucidated. Herein, it is reported that PPK2 class III enzymes from two different bacterial species catalyze the phosphorylation of adenosine mono‐ (AMP) into triphosphate (ATP) not only through step‐by‐step phosphorylation, but also by pyrophosphorylation. These are the first PPK2 enzymes that have been shown to possess polyphosphate‐dependent pyrophosphorylation activity.

A universal polyphosphate kinase: PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate

“…Targeted QQQ-MS/MS measurements with corresponding MRM mass transitions using phosphate ester residues as [M-2H] − fragment ions ( m/z 79) confirmed the additional formation of nucleoside tetraphosphates, namely, adenosine-tetraphosphate (AT(4)P), guanosine-tetraphosphate (GT(4)P), cytidine-tetraphosphate (CT(4)P), desoxy-thymidine-tetraphosphate (dTT(4)P) and uridine-tetraphosphate in all five samples (Online Resources 2 and 5 ). These findings correspond to the previous identification of AT(4)P as an additional reaction product of the PPK of E. coli (Ahn and Kornberg 1990 ), recently also found for PPK2s of Meiothermus ruber (Mordhorst et al 2019 ) and of Delftia tsuruhatensis (Ogawa et al 2019 ). It should be noted that the nucleoside tetraphosphates were only formed when manganese was present in the assay buffer and were not detected in assays with magnesium salts.…”

“…Despite an increasing number of reports on the properties of PPKs and on the utility of PPKs for the regeneration of ATP in ATP-requiring reactions (for recent examples see (Kamatani et al 2018 ; Ogawa et al 2019 ; Zhang et al 2020 ; Wang et al 2020 )), only few PPKs have been tested for their specificity towards pyrimidine nucleotides. The first one is the PPK of E. coli (a PPK1 type enzyme that prefers ADP over GDP and GDP over UDP and CDP (Kuroda and Kornberg 1997 )) and the other is PPK2 of Meiothermus ruber .…”

Characterization of Agrobacterium tumefaciens PPKs reveals the formation of oligophosphorylated products up to nucleoside nona-phosphates

“…Bacteria can have only the PPK1 type of PPKs, only the PPK2 type of PPKs, or both types of PPKs (PPK1 and PPK2) (Rao et al 2009). Several recent reports describe that some PPK2s are able to form nucleotides with four (Mordhorst et al 2019;Ogawa et al 2019;Hildenbrand et al 2019) or even five (Mordhorst et al 2019) phosphate residues.…”

Synthetic Enzyme‐Catalyzed CO2 Fixation Reactions

“…Finally, proof of concept preparative biocatalytic carboxylation reactions simulating conditions often used in industrial processes, such as high substrate loading and incorporating co‐factor recycling systems will greatly enhance the attractiveness of enzymatic carboxylation for industrial exploitation. Recent advances in ATP‐cofactor recycling systems [139–141] can be explored to improve applicability of ATP‐dependent carboxylation processes. It may be necessary to employ green organic co‐solvents or biphasic systems to improve substrate solubility, prevent product inhibition and enhance the performance of the system.…”