Polyphosphate kinase (PPK2), a potent, polyphosphate-driven generator of GTP

Abstract

An enzyme that uses inorganic polyphosphate (poly P) as a donor to convert GDP to GTP has been purified 1,300-fold to homogeneity from lysates of Pseudomonas aeruginosa PAOM5. Poly P chains of 30 -50 residues are optimal; those of 15-700 residues can also serve. GDP is preferred over ADP among nucleoside diphosphate acceptors. This nucleoside diphosphate kinase (NDK) activity resides in the same protein isolated for its synthesis of poly P from GTP and designated PPK2 in an accompanying report. The reaction that synthesizes poly P and the reaction that utilizes poly P differ in their kinetic features. Especially notable is the catalytic potency of the NDK activity, which is 75-fold greater than that of poly P synthesis. PPK2 appears in the stationary phase of growth and reaches NDK levels of 5-10% that of the classic NDK; both kinase activities may figure in the generation of the guanosine precursors in the synthesis of alginate, an exopolysaccharide essential for the virulence of P. aeruginosa.A mong its several functions, inorganic polyphosphate (poly P) serves as a kinase donor to generate ATP and GTP (1-3). Universally distributed in all cells, these chains of phosphate residues, usually hundreds long, linked by phosphoanhydride (metaphosphate) bonds, as in ATP, can be used to phosphorylate glucose, nucleoside diphosphates, and protein (3). In this capacity, poly P was found in extracts of Pseudomonas aeruginosa to be a donor to GDP and a potent source of GTP (4, 5). Of particular physiologic and clinical interest are the sources of GTP needed by P. aeruginosa to provide the intermediates for the synthesis of alginate, its exopolysaccharide associated with mucoidy and virulence (6-8).The present study was undertaken to purify the poly P donor activity designated earlier as PNDK (poly P-driven nucleoside diphosphate kinase; ref. 5) to characterize the enzyme, identify its gene, and, with that, explore the physiologic consequences of overexpression and removal of the gene. At the same time, we were also in pursuit of a putative poly P kinase (PPK2) activity in extracts of P. aeruginosa in addition to the known PPK1 (9, 10) that converts ATP to poly P. The PNDK and PPK2 activities, ostensibly different because of different kinetic features, have proved on isolation of the homogeneous proteins in each case to be the same and to be encoded by the same gene, now designated ppk2 (11). In this report, we describe the isolation and structural properties of PPK2 and its kinetic features based on its activity as a nucleoside diphosphate kinase (NDK). The accompanying report (11) describes the genetic sequence of PPK2 and its conservation in many other bacteria, as well as features of the enzyme in the synthesis of poly P from GTP or ATP. Materials and MethodsStrains and Materials. P. aeruginosa PAOM5 (12), the ⌬ppk::tet derivative of non-mucoid strain PAO1, was used in this study. All of the reagents including poly P 15 were obtained from Sigma. Purified PPK1 was a generous gift from S. Lee (ICOS Corporation, Bothell, ...

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

“…Interestingly, the previously characterized PA0141 has been shown to prefer GDP over ADP as a substrate (12), indicating that short PPK2 proteins can have different substrate preferences (ADP or GDP). PA3455 also exhibited significant activity with dAMP (8.8-9.9 mol/min per mg of protein), dGMP (2.0 mol/min per mg of protein), IMP (2.7-2.8 mol/min per mg of protein), and XMP (1.1-1.6 mol/min per mg of protein).…”

“…tomato) and 6 1-domain PPK2 proteins (PA2428 from P. aeruginosa, SMc02148, SMa0172, and SMa0670 from S. meliloti, Atu0418 from Agrobacterium tumefaciens, and RPA4569 from Rhodopseudomonas palustris). With polyP (polyP [12][13] ) as a phosphodonor, all of the 1-domain PPK2 proteins exhibited polyP-dependent ADP phosphorylation activity and generated ATP (2.3-13.7 mol/min per mg of protein) [supporting information (SI) Fig. S1].…”

“…PPK1 (EC 2.7.4.1; InterPro IPR003414) is responsible for the synthesis of most of the cellular polyP, using the terminal phosphate of ATP as substrate (10,11). The only characterized PPK2 (IPR005660) enzyme, PA0141 from Pseudomonas aeruginosa, uses polyP as a substrate to generate GTP from GDP (12). PPK2 shows no sequence similarity to PPK1 and is distinguished by much higher polyP utilization activity (13).…”

“…PPK2 shows no sequence similarity to PPK1 and is distinguished by much higher polyP utilization activity (13). The PPK2 enzyme PA0141 from P. aeruginosa preferentially phosphorylates GDP to GTP, whereas its affinity for ADP is 2.5 times lower (12,13). Because the expression of PA0141 in P. aeruginosa cells is increased Ͼ100 times during the stationary growth phase, it has been suggested that this enzyme functions in the generation of GTP for the synthesis of alginate, an exopolysaccharide essential for the virulence of P. aeruginosa (12).…”

“…The PPK2 enzyme PA0141 from P. aeruginosa preferentially phosphorylates GDP to GTP, whereas its affinity for ADP is 2.5 times lower (12,13). Because the expression of PA0141 in P. aeruginosa cells is increased Ͼ100 times during the stationary growth phase, it has been suggested that this enzyme functions in the generation of GTP for the synthesis of alginate, an exopolysaccharide essential for the virulence of P. aeruginosa (12). In the social slime mold Dictyostelium discoideum, a different PPK was found (DdPPK2) with sequence and properties similar to that of actin-related proteins (Arps) (14).…”