Functional characterization of Zymomonas mobilis polyphosphate kinase 2 (PPK2); implications for polyphosphate and nucleotide metabolism

Abstract

Inorganic polyphosphate (poly-P) is a linear biopolymer comprised of tens to hundreds of phosphate monomers. Poly-P plays a variety of important physiological roles in all living organisms; for example, helping microbial cells survive and adapt to external stresses. Type 2 polyphosphate kinase (PPK2) enzymes are encoded in a wide variety of bacterial organisms. General consensus is the primary function of PPK2 proteins is to catalyze the phosphorylation of nucleotide monophosphate (NMP) or nucleotide diphosphate (NDP) substrates using polyphosphate as the phosphate donor. However, PPK2 proteins are diverse, and their full range of functions remains to be determined. Here, we report the biochemical characterization of the PPK2 homologue encoded by the ethanol-producing bacterium Zymomonas mobilis (ZM-PPK2). The ZZ6_0566 gene from Zymomonas mobilis subsp. mobilis ATCC 29191, which encodes a putative one-domain PPK2 protein of 261 amino acids, was cloned and overexpressed in Escherichia coli. Size exclusion chromatography indicated that the purified recombinant ZM-PPK2 protein adopts a stable tetrameric arrangement in solution. Its substrate range and biochemical activities were determined by analyzing incubation mixtures using chromatography and gel-based approaches; in conjunction with fluorometric and spectrophotometric assays. Results indicated that purine 5’-monophosphates, e.g. AMP and GMP were efficiently phosphorylated to the corresponding 5’-diphosphates using poly-P as the phosphate donor. Pyrimidine 5’-monophosphates e.g. UMP were not utilized. To a lesser extent, ZM-PPK2 also catalyzed the transfer of phosphate units from poly-P to GDP to form GTP; but did not utilize ADP. Medium and long chain length poly-P substrates were utilized more effectively than short chain lengths, and the reverse reactions (i.e. polyphosphate synthesis) were not catalyzed to notable levels. Mg(II) or Mn(II) ions were essentially required for all activities; which displayed a broad pH optimum (pH 6.8 to 8.8). Studies are ongoing to establish its biological functions. In conclusion, our preliminary results suggest that the primary function of ZM-PPK2 is to utilize medium to long chain polyphosphate for the synthesis of GDP and ADP nucleotides.