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PSI-SGKB [doi:10.1038/fa_psisgkb.2009.1]
Featured Article - February 2009
Short description: Despite the cellular importance of inorganic polyphosphate, particularly in bacteria, exactly how it is processed is not known. Two new polyphosphate kinase structures should make things clearer.Proc Natl Acad Sci USA 105, 17730-17735 (2008)

Inorganic polyphosphate (polyP) is a linear polymer of phosphates linked together by high-energy bonds. It is found in all organisms and has multiple roles: for example, it can replace ATP in kinase reactions and chelate metals. It might have been a key source of energy in the pre-ATP world.

In bacteria, polyP levels are controlled by several polyphosphatases as well as by two families of polyP kinases, PPK1 and PPK2. The sequences of these enzymes are not related. PPK1 synthesizes most of the polyP in the cell, using ATP as a substrate, whereas the only example of PPK2 characterized up to now generates GTP from GDP. The presence of PPK2-like genes in the genomes of many pathogens, and its absence from the human genome, suggest that PPK2s would be good targets for new antibacterial drugs.

Boguslaw Nocek from PSI MCSG and collaborators investigated the structure and function of two PPK2 enzymes, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa. They chose these enzymes after a BLAST search of the InterPro database for genomes possessing genes likely to encode homologs of the known PPK2 enzyme PA0141 from P. aeruginosa. They found that many genomes contained several PPK2 paralogs. Most of the proteins they found had a single PPK2 domain (of approximately 230 residues), but a few contained two fused PPK2 domains. PA3455 has two domains whereas SMc02148 is a one-domain protein.

Preliminary bioinformatic studies suggested that PPK2 belongs to a superfamily of P-loop kinases but forms its own distinctive class that closely resembles nucleotide kinases. P-loop kinases have two conserved sequence motifs, Walker A and Walker B, and contain a lid module.

The authors solved the crystal structures of SMc02148 and PA3455, both of which comprise a three-layer α/β/α sandwich fold with an α-helical lid similar to the structure of microbial thymidylate kinases.

Seeing the structures, the authors realized that the N-terminal domain of PA3455 lacked two important residues in the Walker B motif and so might be inactive. To check this, they cloned the individual N- and C-terminal domains of PA3455 and purified the expressed protein. The C-terminal fragment catalyzed polyP-dependent phosphorylation of AMP to ADP, but the N-terminal PPK2 domain showed no detectable activity. The Walker B Asp–Arg motif plays an important role in the catalytic activity of thymidylate kinases, and also in PKK2.

Nocek et al. used alanine-replacement mutagenesis to identify nine conserved residues needed for activity. These include residues in the Walker B motif and three close to or within the Walker A motif. Mutation of residues in the lid module also produced low or no activity.

Nocek et al. used alanine-replacement mutagenesis to identify nine conserved residues needed for activity. These include residues in the Walker B motif and three close to or within the Walker A motif. Mutation of residues in the lid module also produced low or no activity.

The structural similarity between the PPK2 domains and the thymidylate kinases suggest that these enzymes have a common evolutionary origin and similar catalytic mechanism. The authors propose that in PPK2 enzymes, the carboxylate group on the aspartate in the Walker A motif acts as a general base, attracting a proton from the hydroxyl of the terminal phosphate of the nucleotide and thus preparing it to attack the terminal phosphate of polyP.

This work shows that enzymes of the PPK2 family preferentially function as polyP-dependent nucleotide kinases. Proteins possessing two PPK2 domains, such as PA3455, phosphorylate AMP to ADP, whereas single-domain PPK2 kinases, such as SMc02148, use polyP to phosphorylate ADP or GDP. Acting together, the two groups can convert AMP to ATP. PolyP-driven synthesis of ADP and ATP is likely to be an important survival mechanism for microbial cells under conditions of stress or during infection.

Maria Hodges

References

  1. B. Nocek et al. Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria.
    Proc Natl Acad Sci USA 105, 17730-17735 (2008).

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