Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria Boguslaw Noceka, Samvel Kochinyanb, Michael Proudfootb, Greg Brownb, Elena Evdokimovaa,b, Jerzy Osipiuka, Aled M. Edwardsa,b, Alexei Savchenkoa,b, Andrzej Joachimiaka,c,1, and Alexander F. Yakuninb,1 aMidwest Center for Structural Genomics and Structural Biology Center, Department of Biosciences, Argonne National Laboratory, 9700 South Cass Avenue, Building 202, Argonne, IL 60439; bBanting and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada M5G 1L6; and cDepartment of Biochemistry and Molecular Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637 Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved September 30, 2008 (received for review August 1, 2008) Inorganic polyphosphate (polyP) is a linear polymer of tens or hun- exopolysaccharide essential for the virulence of P. aeruginosa (12). dreds of phosphate residues linked by high-energy bonds. It is found In the social slime mold Dictyostelium discoideum, a different PPK in all organisms and has been proposed to serve as an energy source was found (DdPPK2) with sequence and properties similar to that in a pre-ATP world. This ubiquitous and abundant biopolymer plays of actin-related proteins (Arps) (14). DdPPK2 is a complex of 3 numerous and vital roles in metabolism and regulation in prokaryotes Arps (Arp1, Arp2, and Arpx) and resembles the actin family in and eukaryotes, but the underlying molecular mechanisms for most molecular weight, sequence, and filamentous structure. Remark- activities of polyP remain unknown. In prokaryotes, the synthesis and ably, DdPPK2 can polymerize into an actin-like filament concurrent utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 with the synthesis of polyP (14). Presently, there are 1,380 PPK1- and PPK2, and polyphosphatases. Here, we present structural and like and 722 PPK2-like sequences in the sequenced microbial functional characterization of the PPK2 family. Proteins with a single genome databases (InterPro database; www.ebi.ac.uk/interpro) PPK2 domain catalyze polyP-dependent phosphorylation of ADP to with many genomes containing both PPK1 and PPK2. The PPK1 ATP, whereas proteins containing 2 fused PPK2 domains phosphor- enzymes represent attractive drug targets (13), and the presence of ylate AMP to ADP. Crystal structures of 2 representative proteins, PPK2-like genes in the genomes of a wide spectrum of pathogens SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomo- coupled with their absence in the human genome suggests that nas aeruginosa, revealed a 3-layer ␣/␤/␣ sandwich fold with an PPK2s could also be targets for therapeutic intervention. ␣-helical lid similar to the structures of microbial thymidylate kinases, Here, we present the results of structural and biochemical suggesting that these proteins share a common evolutionary origin characterization of 2 groups of PPK2 proteins containing 1 or 2 and catalytic mechanism. Alanine replacement mutagenesis identi- fused PPK2 domains. We have demonstrated that the first group, fied 9 conserved residues, which are required for activity and include which contains a single PPK2 domain, catalyzes the polyP- the residues from both Walker A and B motifs and the lid. Thus, the dependent phosphorylation of nucleoside diphosphates (ADP, PPK2s represent a molecular mechanism, which potentially allow GDP) to nucleoside triphosphates, whereas the proteins with 2 bacteria to use polyP as an intracellular energy reserve for the fused PPK2 domains phosphorylate nucleoside monophosphates generation of ATP and survival. (AMP, GMP) to the respective diphosphates. Crystal structures of 2 representative proteins were solved, and site-directed mutagen- crystal structure ͉ mutagenesis ͉ Walker motif ͉ AMP phosphorylation ͉ esis identified the conserved residues important for catalysis. ADP phosphorylation Results and Discussion norganic polyphosphate (polyP) is a linear polymer composed of Two Groups of PPK2 Enzymes. A BLAST search of the sequenced genomes using the sequence of the biochemically characterized Itens to hundreds of orthophosphate residues (Pi) linked by the energy-rich phosphoanhydride bonds, and it is found in all pro- PPK2 enzyme PA0141 from P. aeruginosa as a query revealed that karyotes and eukaryotes (1–3). PolyP has numerous biological many genomes encode 2 or 3 PPK2 paralogs, most of which contain functions that include substitution for ATP in kinase reactions, a single PPK2 domain Ϸ230 residues in length (1-domain PPK2). acting as an energy source or storage reservoir of Pi, chelating In addition, some genomes show the presence of a longer gene metals, and adjusting cellular physiology during growth, develop- (496–544 residues) with 2 fused PPK2 domains, perhaps produced ment, stress, starvation, and virulence (1, 4). Bacteria with low by a gene duplication event (2-domain PPK2). For example, the intracellular polyP levels show defective responses to various stress genome of P. aeruginosa encodes 2 1-domain PPK2 (PA0141 and conditions, biofilm formation, quorum sensing, motility, and other PA2428) and 1 2-domain PPK2 (PA3455) proteins, whereas Sino- virulence properties (3, 5). Depending on the physiological state of rhizobium meliloti has 3 genes encoding only 1-domain PPK2 the bacterium, the intracellular concentration of polyP is 0.1–200 proteins (SMc02148, SMa0172, and SMa0670). Sequence analysis mM. The polyP level is controlled mainly by the activity of several of 2-domain PPK2 proteins revealed that their C-terminal domain polyphosphatases and 2 families of polyP kinases, PPK1 and PPK2 is more conserved and shows higher similarity to the sequences of (1, 6–9). PPK1 (EC 2.7.4.1; InterPro IPR003414) is responsible for the Author contributions: B.N., A.M.E., A.S., and A.F.Y. designed research; B.N., S.K., M.P., G.B., synthesis of most of the cellular polyP, using the terminal phosphate E.E., J.O., and A.F.Y. performed research; B.N., S.K., M.P., G.B., E.E., J.O., A.M.E., A.S., A.J., of ATP as substrate (10, 11). The only characterized PPK2 and A.F.Y. analyzed data; and B.N., A.M.E., A.S., A.J., and A.F.Y. wrote the paper. (IPR005660) enzyme, PA0141 from Pseudomonas aeruginosa, uses The authors declare no conflict of interest. polyP as a substrate to generate GTP from GDP (12). PPK2 shows This article is a PNAS Direct Submission. no sequence similarity to PPK1 and is distinguished by much higher Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, polyP utilization activity (13). The PPK2 enzyme PA0141 from P. www.pdb.org (PDB ID codes 3CZP and 3CZQ). aeruginosa preferentially phosphorylates GDP to GTP, whereas its 1To whom correspondence may be addressed. E-mail: [email protected] or a.iakounine@ affinity for ADP is 2.5 times lower (12, 13). Because the expression utoronto.ca. of PA0141 in P. aeruginosa cells is increased Ͼ100 times during the This article contains supporting information online at www.pnas.org/cgi/content/full/ stationary growth phase, it has been suggested that this enzyme 0807563105/DCSupplemental. functions in the generation of GTP for the synthesis of alginate, an © 2008 by The National Academy of Sciences of the USA 17730–17735 ͉ PNAS ͉ November 18, 2008 ͉ vol. 105 ͉ no. 46 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807563105 Downloaded by guest on September 30, 2021 Fig. 1. Structure-based sequence align- ment of the PPK2 domains of the proteins containing 1 or 2 PPK2 domains. Residues conserved in all PPK2 proteins are high- lighted 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 aster- isks, and the lid module is indicated by the BIOCHEMISTRY thick line. The single PPK2 domain pro- teins (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 do- main; PA3455-C, C-terminal PPK2 domain), and PSPTO1640 (Q886D9; PSPTO1640-N, N- terminal domain; PSPTO1640-C, C-terminal domain). 1-domain PPK2 proteins than their N-terminal domains (Fig. 1). of protein) [supporting information (SI) Fig. S1]. In contrast, both Previous bioinformatics analysis indicated that PPK2 proteins 2-domain PPK2 proteins catalyzed the polyP-dependent phosphor- belong to the large superfamily of P-loop kinases and represent a ylation of AMP and produced ADP as a final product (39.0–40.0 distinct family of predicted kinases closely related to nucleotide ␮mol/min per mg of protein) (Fig. S1). Recently, the polyP:AMP kinases (15). P-loop kinases are characterized by the presence of 2 phosphotransferase PAP from Acinetobacter johnsonii 210A has conserved sequence motifs (Walker A and Walker B) and the lid been shown to phosphorylate AMP in a polyP-dependent reaction (16). module. The Walker A motif (or P-loop, GXXXXGK) binds the ␤- This protein shares 40% sequence identity with PA3455 and contains and ␥-phosphates of ATP, whereas the conserved Asp of the 2 fused PPK2 domains, indicating that it is a 2-domain PPK2 protein. Walker B motif coordinates a Mg2ϩ cation, which is also bound to Both groups of PPK2 proteins were also capable of phosphory- the ␤- and ␥-phosphates of ATP (15). lating GMP to GDP (PA3455 and PSPTO1640) or GDP to GTP (SMc02148, SMa0172, and SMa0670), but this activity was Ϸ3 times Enzymatic Activity of Purified PPK2 Proteins. We overexpressed in lower than that with adenosine nucleotides (Table 1, data shown for Escherichia coli and purified 2 2-domain PPK2 proteins (PA3455 PA3455 and SMc02148). Interestingly, the previously characterized from P. aeruginosa and PSPTO1640 from P. syringae pv. tomato) PA0141 has been shown to prefer GDP over ADP as a substrate and 6 1-domain PPK2 proteins (PA2428 from P.