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A new subfamily of polyphosphate kinase 2 (class III PPK2) catalyzes both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation.

ABSTRACT: Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (Pi) residues linked by "high-energy" phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities.

SUBMITTER: Motomura K

PROVIDER: S-EPMC3993171 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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A new subfamily of polyphosphate kinase 2 (class III PPK2) catalyzes both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation.

Motomura Kei K Hirota Ryuichi R Okada Mai M Ikeda Takeshi T Ishida Takenori T Kuroda Akio A

Applied and environmental microbiology 20140214 8

Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (Pi) residues linked by "high-energy" phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under condi ...[more]

PMID: 24532069

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Project description:In plants, the biosynthesis of isopentenyl diphosphate, the central precursor of all isoprenoids, proceeds via two separate pathways. The cytosolic compartment harbors the mevalonate pathway, whereas the newly discovered deoxyxylulose 5-phosphate pathway, which also operates in certain eubacteria, including Escherichia coli, is localized to plastids. Only the first two steps of the plastidial pathway, which involve the condensation of pyruvate and glyceraldehyde 3-phosphate to deoxyxylulose 5-phosphate followed by intramolecular rearrangement and reduction to 2-C-methylerythritol 4-phosphate, have been established. Here we report the cloning from peppermint (Mentha x piperita) and E. coli, and expression, of a kinase that catalyzes the phosphorylation of isopentenyl monophosphate as the last step of this biosynthetic sequence to isopentenyl diphosphate. The plant gene defines an ORF of 1,218 bp that, when the proposed plastidial targeting sequence is excluded, corresponds to approximately 308 aa with a mature size of approximately 33 kDa. The E. coli gene (ychB), which is located at 27.2 min of the chromosomal map, consists of 852 nt, encoding a deduced enzyme of 283 aa with a size of 31 kDa. These enzymes represent a conserved class of the GHMP family of kinases, which includes galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase, with homologues in plants and several eubacteria. Besides the preferred substrate isopentenyl monophosphate, the recombinant peppermint and E. coli kinases also phosphorylate isopentenol, and, much less efficiently, dimethylallyl alcohol, but dimethylallyl monophosphate does not serve as a substrate. Incubation of secretory cells isolated from peppermint glandular trichomes with isopentenyl monophosphate resulted in the rapid production of monoterpenes and sesquiterpenes, confirming that isopentenyl monophosphate is the physiologically relevant, terminal intermediate of the deoxyxylulose 5-phosphate pathway.

Project description:The Mycobacterium tuberculosis gene Rv3232c/MT3329 (ppk2) encodes a class II polyphosphate kinase, which hydrolyzes inorganic polyphosphate (poly P) to synthesize GTP. We assessed the role of ppk2 in M. tuberculosis poly P regulation, antibiotic tolerance, and virulence. A ppk2-deficient mutant (ppk2::Tn) and its isogenic wild-type (WT) and complemented (Comp) strains were studied. For each strain, the intrabacillary poly P content, MIC of isoniazid, and growth kinetics during infection of J774 macrophages were determined. Multiplex immunobead assays were used to evaluate cytokines elaborated during macrophage infection. The requirement of ppk2 for M. tuberculosis virulence was assessed in the murine model. The ppk2::Tn mutant was found to have significantly increased poly P content and a 4-fold increase in the MIC of isoniazid relative to the WT and Comp strains. The ppk2::Tn mutant showed reduced survival at day 7 in activated and naive J774 macrophages relative to the WT. Naive ppk2::Tn mutant-infected macrophages showed increased expression of interleukin 2 (IL-2), IL-9, IL-10, IL-12p70, and gamma interferon (IFN-?) relative to WT-infected macrophages. The ppk2::Tn mutant exhibited significantly lower lung CFU during acute murine infection compared to the control groups. ppk2 is required for control of intrabacillary poly P levels and optimal M. tuberculosis growth and survival in macrophages and mouse lungs. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a highly successful human pathogen because it has developed mechanisms to multiply and survive in the lungs by circumventing the immune system. Identification of virulence factors responsible for M. tuberculosis growth and persistence in host tissues may assist in the development of novel strategies to treat TB. In this study, we found that the mycobacterial enzyme polyphosphate kinase 2 (PPK2) is required for controlling intracellular levels of important regulatory molecules and for maintaining susceptibility to the first-line anti-TB drug isoniazid. In addition, PPK2 was found to be required for M. tuberculosis growth in the lungs of mice, at least in part by suppressing the expression of certain key cytokines and chemokines by inactivated lung macrophages.

Dataset Information

A new subfamily of polyphosphate kinase 2 (class III PPK2) catalyzes both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation.

Publications

A new subfamily of polyphosphate kinase 2 (class III PPK2) catalyzes both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation.

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