Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria

Abstract

Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer ␣/␤/␣ sandwich fold with an ␣-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.crystal structure ͉ mutagenesis ͉ Walker motif ͉ AMP phosphorylation ͉ ADP phosphorylation I norganic polyphosphate (polyP) is a linear polymer composed of tens to hundreds of orthophosphate residues (P i ) linked by the energy-rich phosphoanhydride bonds, and it is found in all prokaryotes and eukaryotes (1-3). PolyP has numerous biological functions that include substitution for ATP in kinase reactions, acting as an energy source or storage reservoir of P i , chelating metals, and adjusting cellular physiology during growth, development, stress, starvation, and virulence (1, 4). Bacteria with low intracellular polyP levels show defective responses to various stress conditions, biofilm formation, quorum sensing, motility, and other virulence properties (3,5). Depending on the physiological state of the bacterium, the intracellular concentration of polyP is 0

Ppn2, a novel Zn2+-dependent polyphosphatase in the acidocalcisome-like yeast vacuole

“…Substantial stores of polyP exist in prokaryotic and eukaryotic microorganisms. PolyP metabolism is tightly integrated into the overall energy metabolism of the cell and polyP is used by the cells as a P i reserve and in phosphorylation and polyphosphorylation reactions (Azevedo et al, 2015;Livermore et al, 2016;Nocek et al, 2008;Rao et al, 2009). PolyP is important in detoxification of heavy metals and in counteracting alkaline and osmotic stress (Keasling, 1997;Pick and Weiss, 1991;Rohloff and Docampo, 2008).…”

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

“…Metagenomic analysis of Accumulibacter phosphatis, a dominant polyP-accumulating microorganism in the enhanced biological phosphorus removal (EBPR) system, revealed the presence of five paralogs of PPK2 (12), suggesting that metabolism of polyP by PPK2 is important for its survival in the EBPR system. Nocek et al found that most of the PPK2 enzymes contain a single domain of ϳ230 amino acids in length (1-domain PPK2), while some contain two homologous domains (2-domain PPK2), a probable result of a gene duplication event (13). In addition, they demonstrated that 1-domain PPK2 catalyzes nucleoside triphosphate (ATP and GTP) synthesis from the respective nucleoside diphosphates and polyP, whereas the 2-domain PPK2 catalyzes nucleoside diphosphate (ADP and GDP) synthesis from the respective nucleoside monophosphates and polyP (13).…”

Polyphosphate Deficiency in Mycobacterium tuberculosis Is Associated with Enhanced Drug Susceptibility and Impaired Growth in Guinea Pigs

“…In bacteria, some of the enzymes involved in polyP metabolism are polyphosphate kinase 1 (PPK-1), which catalyzes the reversible transfer of the terminal (␥) phosphate of ATP to form polyP, and polyphosphatase (PPX), which processively hydrolyzes the terminal residues of polyP to liberate P i (21)(22)(23). Polyphosphate kinase 2 (PPK-2) is another enzyme involved in polyP metabolism that drives synthesis of GTP and ATP using polyP as a phosphate donor (24)(25)(26).…”