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

Frank, Celina; Teleki, Attila; Jendrossek, Dieter

doi:10.1007/s00253-020-10891-7

Cited by 12 publications

(8 citation statements)

References 29 publications

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“…About 5% AMP was observed during the reaction, which is derived from the starting material and is not further accumulating over the course of the reaction. Higher phosphorylated compounds such as adenosine 5'-tetraphosphate, which are additional reaction products of PPK2 catalysed reactions, were not detected under the conditions applied, this usually requires higher enzyme concentrations [ 7 – 8 ]. A similar equilibrium concentration was observed for Vc PPK1 (ADP/ATP 30%:70%, Figure S1, Supporting Information File 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…PPK2 were later subdivided into three classes: PPK2-I, PPK2-II, and PPK2-III phosphorylating nucleotide diphosphates (NDPs), nucleotide monophosphates (NMPs), and both, respectively [ 6 ]. Nevertheless, these substrate profiles rather seem to be preferences, as most enzymes catalyse all phosphorylation steps during extended reaction times; also higher phosphorylated species have been detected in the reactions [ 7 – 8 ]. The enzymes characterised from E. coli ( Ec PPK1) and Sinorhizobium meliloti (renamed Ensifer meliloti, Sm PPK2) are often regarded as model enzymes for PPK1 and PPK2 [ 9 – 10 ].…”

Section: Introductionmentioning

confidence: 99%

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Make or break: the thermodynamic equilibrium of polyphosphate kinase-catalysed reactions

Keppler¹,

Moser²,

Jessen³

et al. 2022

Beilstein J. Org. Chem.

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Polyphosphate kinases (PPKs) have become popular biocatalysts for nucleotide 5'-triphosphate (NTP) synthesis and regeneration. Two unrelated families are described: PPK1 and PPK2. They are structurally unrelated and use different catalytic mechanisms. PPK1 enzymes prefer the usage of adenosine 5'-triphosphate (ATP) for polyphosphate (polyP) synthesis while PPK2 enzymes favour the reverse reaction. With the emerging use of PPK enzymes in biosynthesis, a deeper understanding of the enzymes and their thermodynamic reaction course is of need, especially in comparison to other kinases. Here, we tested four PPKs from different organisms under the same conditions without any coupling reactions. In comparison to other kinases using phosphate donors with comparably higher phosphate transfer potentials that are characterised by reaction yields close to full conversion, the PPK-catalysed reaction reaches an equilibrium in which about 30% ADP is left. These results were obtained for PPK1 and PPK2 enzymes, and are supported by theoretical data on the basic reaction. At high concentrations of substrate, the different kinetic preferences of PPK1 and PPK2 can be observed. The implications of these results for the application of PPKs in chemical synthesis and as enzymes for ATP regeneration systems are discussed.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Make or break: the thermodynamic equilibrium of polyphosphate kinase-catalysed reactions

Keppler¹,

Moser²,

Jessen³

et al. 2022

Beilstein J. Org. Chem.

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show abstract

“…Analytical preparation of sample extracts and chromatographic separation of nonderivatized polar metabolites by alkaline polymer-based zwitterionic hydrophilic interaction chromatography (ZIC-pHILIC) were performed as previously described [ 105 , 106 ]. Defined standard mixtures and samples with adapted dilution containing 50 μM 2-keto-3-deoxy-6-phosphogluconate (KDPG) and α-amino isobutyric acid (AIBA) as global internal standard (measurement) were injected (5 μL) into a Sequant ZIC-pHILIC column (150 × 2.1 mm, 5 μm, Merck Millipore, Darmstadt, Germany) equipped with a guard column (20 × 2.1 mm, 5 μm, Merck Millipore, Darmstadt, Germany) maintained at 40 °C.…”

Section: Methodsmentioning

confidence: 99%

Monitoring Intracellular Metabolite Dynamics in Saccharomyces cerevisiae during Industrially Relevant Famine Stimuli

et al. 2022

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Carbon limitation is a common feeding strategy in bioprocesses to enable an efficient microbiological conversion of a substrate to a product. However, industrial settings inherently promote mixing insufficiencies, creating zones of famine conditions. Cells frequently traveling through such regions repeatedly experience substrate shortages and respond individually but often with a deteriorated production performance. A priori knowledge of the expected strain performance would enable targeted strain, process, and bioreactor engineering for minimizing performance loss. Today, computational fluid dynamics (CFD) coupled to data-driven kinetic models are a promising route for the in silico investigation of the impact of the dynamic environment in the large-scale bioreactor on microbial performance. However, profound wet-lab datasets are needed to cover relevant perturbations on realistic time scales. As a pioneering study, we quantified intracellular metabolome dynamics of Saccharomyces cerevisiae following an industrially relevant famine perturbation. Stimulus-response experiments were operated as chemostats with an intermittent feed and high-frequency sampling. Our results reveal that even mild glucose gradients in the range of 100 µmol·L−1 impose significant perturbations in adapted and non-adapted yeast cells, altering energy and redox homeostasis. Apparently, yeast sacrifices catabolic reduction charges for the sake of anabolic persistence under acute carbon starvation conditions. After repeated exposure to famine conditions, adapted cells show 2.7% increased maintenance demands.

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“…Similarly, the class III PPK2 from Delftia tsuruhatensis also makes AP4 [ 51 ]. Most recently, the class I A. tumefaciens PPK2 has been shown to catalyze the formation of oligophosphorylated nucleosides from GDP, CDP, dTDP, and UDP, with nona-phosphorylated adenosine being the largest product observed [ 45 ]. Crystallization of Ft PPK2 with AMP-PCP and polyP resulted in a structure that clearly showed two extra phosphate groups added to AMP-PCP to yield AMP-PCPPP ( Figure 2 ), though this species could not be detected in free solution enzyme reactions [ 53 ].…”

Section: Ppk2 Enzymologymentioning

confidence: 99%

Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence

Neville

Roberge

Jia

2022

IJMS

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Inorganic polyphosphate (polyP) has been implicated in an astonishing array of biological functions, ranging from phosphorus storage to molecular chaperone activity to bacterial virulence. In bacteria, polyP is synthesized by polyphosphate kinase (PPK) enzymes, which are broadly subdivided into two families: PPK1 and PPK2. While both enzyme families are capable of catalyzing polyP synthesis, PPK1s preferentially synthesize polyP from nucleoside triphosphates, and PPK2s preferentially consume polyP to phosphorylate nucleoside mono- or diphosphates. Importantly, many pathogenic bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii encode at least one of each PPK1 and PPK2, suggesting these enzymes may be attractive targets for antibacterial drugs. Although the majority of bacterial polyP studies to date have focused on PPK1s, PPK2 enzymes have also begun to emerge as important regulators of bacterial physiology and downstream virulence. In this review, we specifically examine the contributions of PPK2s to bacterial polyP homeostasis. Beginning with a survey of the structures and functions of biochemically characterized PPK2s, we summarize the roles of PPK2s in the bacterial cell, with a particular emphasis on virulence phenotypes. Furthermore, we outline recent progress on developing drugs that inhibit PPK2 enzymes and discuss this strategy as a novel means of combatting bacterial infections.

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

Cited by 12 publications

References 29 publications

Make or break: the thermodynamic equilibrium of polyphosphate kinase-catalysed reactions

Make or break: the thermodynamic equilibrium of polyphosphate kinase-catalysed reactions

Monitoring Intracellular Metabolite Dynamics in Saccharomyces cerevisiae during Industrially Relevant Famine Stimuli

Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence

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