Polyphosphate:AMP Phosphotransferase and Polyphosphate:ADP Phosphotransferase Activities of Pseudomonas aeruginosa

doi:10.1006/bbrc.2001.4415

Biochemical and Biophysical Research Communications

Volume 281, Issue 3, 2 March 2001, Pages 821-826

https://doi.org/10.1006/bbrc.2001.4415 Get rights and content

Abstract

In Pseudomonas aeruginosa PAO1, we have found massive polyphosphate:AMP phosphotransferase activity and polyphosphate:ADP phosphotransferase activity known as the reverse catalytic activity of polyphosphate kinase which participates in polyphosphate synthesis in the bacterium. Biochemical analysis using the partially purified polyphosphate:ADP phosphotransferase has revealed that it is independent of polyphosphate kinase and can function as polyphosphate-dependent nucleoside diphosphate kinase which most prefers GDP to the other three nucleoside diphosphates as a phospho-acceptor. It has been also demonstrated that polyphosphate:AMP phosphotransferase activity marked in the bacterium mainly originates from the combined action of the polyphosphate:ADP phosphotransferase described above and adenylate kinase. Both of the polyphosphate-utilizing activities require short polyP as a phospho-donor whose chain length is <75.

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Cited by (26)

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Cytidine 5'-monophosphate (5'-CMP), a key intermediate for the production of nucleotide derivatives, has been extensively used in food, agriculture, and medicine industries. Compared to RNA degradation and chemical synthesis, the biosynthesis of 5'-CMP has attracted wide attention due to its relatively low cost and eco-friendliness. In this study, we developed a cell-free regeneration of ATP based on polyphosphate kinase 2 (PPK2) to manufacture 5'-CMP from cytidine (CR). McPPK2 from Meiothermus cerbereus exhibited high specific activity (128.5 U/mg) and was used to accomplish ATP regeneration. McPPK2 and LhUCK (a uridine-cytidine kinase from Lactobacillus helveticus) were combined to convert CR to 5'-CMP. Further, the degradation of CR was inhibited by knocking out cdd from the Escherichia coli genome to enhance 5'-CMP production. Finally, the cell-free system based on ATP regeneration maximized the titer of 5'-CMP up to 143.5 mM. The wider applicability of this cell-free system was demonstrated in the synthesis of deoxycytidine 5'-monophosphate (5'-dCMP) from deoxycytidine (dCR) by incorporating McPPK2 and BsdCK (a deoxycytidine kinase from Bacillus subtilis). This study suggests that the cell-free regeneration of ATP based on PPK2 has the advantage of great flexibility for producing 5'-(d)CMP and other (deoxy)nucleotides.
Catalytic activity profile of polyP:AMP phosphotransferase from Myxococcus xanthus
2021, Journal of Bioscience and Bioengineering
Citation Excerpt :
We next analyzed the effect of polyP chain length on Pap activity. It was shown that A. johnsonii Pap had the highest activity with polyPs containing 18–44 phosphate residues and could not use pyrophosphate, polyP3, and polyP4 (12), whereas P. aeruginosa Pap used polyP15 but had very low activity with pyrophosphate, polyP4, polyP75, and polyP750 as phosphoryl donors (13). M. xanthus Pap could phosphorylate AMP to ADP using most of the tested polyP substrates (polyP4, polyP10-15, polyP60-70, polyP700-1000, and metaphosphate) except pyrophosphate and polyP3 (data not shown), suggesting its wide specificity for phosphoryl donors.
Myxococcus xanthus generates polyphosphates (polyPs) during starvation and forms fruiting bodies through the activity of polyphosphate kinase (Ppk). M. xanthus polyP:AMP phosphotransferase (Pap), a class II Ppk2, catalyzes the transfer of the terminal phosphate from polyP to AMP to yield ADP, but its enzymatic properties have not been investigated in detail. In this study, we found that Pap was activated by Mn²⁺ or Mg²⁺ and required higher concentrations of these ions in reactions with longer polyPs to demonstrate maximum activity. The K_m of Pap for polyP_700-1000 was significantly lower than that for shorter polyPs, but the highest catalytic constant (k_cat) was observed for polyP_60-70. When Pap was incubated with polyP_60-70 and AMP for 3 h, it first generated ADP and then gradually produced ATP, suggesting that M. xanthus Pap also has polyP:ADP phosphotransferase activity similar to that of class III Ppk2 enzymes. During starvation, the specific activity of Pap in M. xanthus was increased by 2.3–2.4-fold at days 1 and 2 of incubation. In addition, recombinant Pap in combination with M. xanthus recombinant enzymes Ppk1 or adenylate kinase (AdkA) could generate ATP from AMP and polyP_60-70. These results suggest a functional role of Pap during M. xanthus starvation, when it might act in cooperation with Ppk1 and/or AdkA to produce ATP from AMP, ADP, and polyP.
Different chain length specificity among three polyphosphate quantification methods
2008, Analytical Biochemistry
Citation Excerpt :
For example, in E. coli, long-chain poly P (but not short-chain poly P) was necessary for enhanced degradation of free ribosomal proteins by the Lon protease [24] responding to amino acid starvation [25], although both were capable of interacting with ribosomes to control translation [26]. In the case of poly P/AMP (adenosine monophosphate) phosphotransferase and poly P/ADP phosphotransferase in Pseudomonas aeruginosa, poly P shorter than 75 residues functioned as a phosphodoner, whereas the longer one was ineffective [27]. S. cerevisiae mutants of the endopolyphosphate (PPN), the enzyme that produces short-chain poly P from the longer one [28], accumulated long-chain poly P and lacked short-chain poly P [29,30].
Polyphosphate is ubiquitous among living organisms and has a variety of biochemical functions. Arbuscular mycorrhizal fungi have been known to accumulate polyphosphate as a key compound for their function. However, an enzymatic assay using polyphosphate kinase (PPK) reverse reaction, in which polyphosphate is converted to adenosine triphosphate (ATP) and quantified by luciferase assay, failed to detect accumulation of polyphosphate in some mycorrhizal root. When yeast exopolyphosphatase (PPX) was applied to these samples, a much higher polyphosphate level was detected than when the PPK assay was applied. Detailed analysis of substrate chain length specificity of these methods using polyphosphate chain length standards revealed that the PPX method was the most appropriate to detect short-chain polyphosphate. The average chain length of the shortest polyphosphate fraction that could be quantified with more than 50% efficiency was 3 for the PPX method and 38 for the PPK method. It was also suggested that the ratio of the PPK value to the PPX value may be useful as a simple and relative index to compare polyphosphate chain length distribution in different samples.
Microbial phosphate removal and polyphosphate production from wastewaters
2003, Advances in Applied Microbiology
Citation Excerpt :
This enzyme is proposed to catalyze the polyphosphate-dependent phosphorylation of nucleoside diphosphates such as ADP (“reverse” PPK activity): Its existence in P. aeruginosa as an activity distinct from that of the reverse reaction of PPK has very recently been proposed (Ishige and Noguchi, 2001). Although the enzyme has not been purified it appears to require short-chain polyP (<75) as the phospho-donor and to have an almost 3-fold higher specificity for guauosine diphosphate (GDP) than ADP as phospho-acceptor [it was speculated that large quantities of guanosine triphosphate (GTP) might be required by this pathogenic organism for exopolysaccharide production].
This chapter focuses on the physiological and biochemical bases of enhanced biological phosphorus (P) removal (EBPR) by microorganisms; this process is characterized by the exposure of activated sludge to periods with and without aeration: the “aerobic” and “anaerobic” phases. In the aerobic stage of EBPR, the microorganisms containing stored poly-β-hydroxybutyrate (PHB)/poly-β-hydroxyvalerate (PHV) replenish their internal polyP reserves, and in doing so, they take up not only the phosphate released during the anaerobic phase of the process but also nearly all the available phosphate from the surrounding environment. The selective enrichment of microorganisms capable of polyP accumulation through the cycling of activated sludge between anaerobic and aerobic zones is, therefore, the basis of conventional EBPR technology. Under favorable conditions, EBPR plants can remove 80–90% of influent phosphate, achieving residual P levels in effluent of less than 1 mg/liter and producing sludges with P content of up to 15%.
Non-canonical nucleosides: Biomimetic triphosphorylation, incorporation into mRNA and effects on translation and structure
2023, FEBS Journal
Functional analysis of polyphosphate in Myxococcus xanthus
2023, Research Square

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^☆: Abbreviations used: ADK, adenylate kinase; NDK, nucleoside diphosphate kinase; PADP, polyphosphate:ADP phosphotransferase; PAP, polyphosphate:AMP phosphotransferase; PEI-TLC, polyethyleneimine-cellulose thin layer chromatography; polyP, inorganic polyphosphate; PPK, polyphosphate kinase; PVDF, polyvinylidene difluoride.

¹: To whom correspondence should be addressed. Fax: 81-479-22-9865. E-mail: [email protected].

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Regular ArticlePolyphosphate:AMP Phosphotransferase and Polyphosphate:ADP Phosphotransferase Activities of Pseudomonas aeruginosa☆

Abstract

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Protein Expression Purif.

Regular Article
Polyphosphate:AMP Phosphotransferase and Polyphosphate:ADP Phosphotransferase Activities of Pseudomonas aeruginosa☆