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. 2008 Nov 18;105(46):17730-5.
doi: 10.1073/pnas.0807563105. Epub 2008 Nov 10.

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

Boguslaw Nocek  1 , Samvel Kochinyan, Michael Proudfoot, Greg Brown, Elena Evdokimova, Jerzy Osipiuk, Aled M Edwards, Alexei Savchenko, Andrzej Joachimiak, Alexander F Yakunin
Affiliations

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

Boguslaw Nocek et al. Proc Natl Acad Sci U S A. .

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 alpha/beta/alpha sandwich fold with an alpha-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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structure-based sequence alignment of the PPK2 domains of the proteins containing 1 or 2 PPK2 domains. Residues conserved in all PPK2 proteins are highlighted in black. The secondary structure elements of the PA3455 C-terminal PPK2 domain and SMc02148 are shown above and below the alignment, respectively. The Walker A motif is designated by balls, the Walker B motif is indicated by asterisks, and the lid module is indicated by the thick line. The single PPK2 domain proteins (short PPK2) comprise SMc02148 (Q92SA6), PA2148 (Q9I154), PA0141 (Q9I6Z1), SMa0670 (Q92ZU4), and SMa0172 (Q930V2). The 2 PPK2 domain proteins (long PPK2) include PA3455 (Q9HYF1; PA3455-N, N-terminal PPK2 domain; PA3455-C, C-terminal PPK2 domain), and PSPTO1640 (Q886D9; PSPTO1640-N, N-terminal domain; PSPTO1640-C, C-terminal domain).
Fig. 2.
Fig. 2.
Overall crystal structures of the PA3455 dimer (A) and SMc02148 tetramer (B). For both proteins, 2 views related by a 90° rotation are shown. PPK2 domains are shown in different colors, and the Roman numerals designate protein monomers. The proteins show a very similar packing with the dimensions 88 × 68 × 50 Å (PA3455) and 85 × 67 × 63 Å (SMc02148).
Fig. 3.
Fig. 3.
Structures of the PPK2 domains of PA3455 and SMc02148. (Upper) Ribbon diagrams of the N-terminal PPK2 domain of PA3455 (A), C-terminal PPK2 domain of PA3455 (B), and SMc02148 monomer (C). The secondary structure elements are shown in different colors (α-helices, dark magenta; β-strands, dark blue; loops, light blue) and are labeled. The Walker A loop is colored in red, the Walker B loop in green, and the lid module helices in dark green, and they are indicated by the capital letters (A, B, and L, respectively). Two yellow dots in 4A designate the boundaries of the disordered part of the lid module of the PA3455 N-terminal PPK2 domain. (Lower) Surface charge distribution of the same PPK2 domains showing the presence of the extended positively charged patch on the left side of the PA3455 C-terminal PPK2 domain (B) and SMc02148 (C), which is absent in the PA3455 N-terminal domain (A). The surface charge distribution was determined by using PyMOL (http://pymol.sourceforge.net).
Fig. 4.
Fig. 4.
Alanine replacement mutagenesis of the PA3455 C-terminal PPK2 domain (A) and SMc02148 (B). Enzymatic activity of purified mutant proteins was measured as polyP-dependent phosphorylation of AMP to ADP (for the C-terminal PPK2 domain of PA3455, residues 261–496) or ADP to ATP (for SMc02148). Experimental conditions were as described in Materials and Methods.
Fig. 5.
Fig. 5.
Close-up stereoview of the active sites of PA3455 and Mc02148. (A) The PA3455 N-terminal domain. (B) The PA3455 C-terminal domain. (C) SMc02148. The secondary structure elements are shown in light gray with the colored catalytic modules: the Walker A loop in violet, the Walker B loop in green, and the lid in teal. The side chains of the conserved residues are shown as orange sticks and labeled.
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