Polyphosphate kinase 2: a modulator of nucleoside diphosphate kinase activity in mycobacteria

Abstract

SummaryMycobacteria encode putative class II polyphosphate kinases (PPKs). We report that recombinant PPK2 of Mycobacterium tuberculosis catalyses the synthesis of GTP from GDP using polyphosphate rather than ATP as phosphate donor. Unlike that of PPK1, this is the favoured reaction of PPK2. The sites of autophosphorylation, H115 and H247, as well as G74 were critical for GTP-synthesizing activity. Compromised survival of a ppk2 knockout (PPK2-KO) of Mycobacterium smegmatis under heat or acid stress or hypoxia, and the ability of ppk2 of M. tuberculosis to complement this, confirmed that PPK2 plays a role in mycobacterial survival under stress. Intracellular ATP : GTP ratio was higher in PPK2-KO compared with the wildtype M. smegmatis, bringing to light a role of PPK2 in regulating the intracellular nucleotide pool. We present evidence that PPK2 does so by interacting with nucleoside diphosphate kinase (Ndk). Pull-down assays and analysis by surface plasmon resonance demonstrated that the interaction requires G74 of PPK2 MTB and 109 LET 111 of NdkMTB. In summary, we unravel a novel mechanism of regulation of nucleotide pools in mycobacteria. Downregulation of ppk2 impairs survival of M. tuberculosis in macrophages, suggesting that PPK2 plays an important role in the physiology of the bacteria residing within macrophages.

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

“…The first characterized P. aeruginosa PPK2 belongs to the class I subfamily. Twelve proteins of the class I subfamily have been characterized and are known to catalyze nucleoside diphosphate phosphorylation (nucleoside triphosphate synthesis) (11,13,(16)(17)(18). On the other hand, the first characterized PPK2 catalyzing nucleoside monophosphate phosphorylation, known as polyP-AMP phosphotransferase (PAP), of A. johnsonii (13), belongs to the class II subfamily.…”

“…While we did not investigate the functions of class III PPK2, many researchers have concluded that polyP-driven nucleotide phosphorylation is important for the survival of microbial cells under conditions of stress or stationary phase (11,17,29). For instance, ppk2 knockout in Mycobacterium species affects intracellular nucleotide pools and impairs cell survival in macrophages (17).…”

Polyphosphate Deficiency in Mycobacterium tuberculosis Is Associated with Enhanced Drug Susceptibility and Impaired Growth in Guinea Pigs

“…In another study, it has been shown that downregulation of ppk-1 in M. tuberculosis using the IPTG (isopropyl-␤-D-thiogalactopyranoside) antisense-inducible system results in a bacteriostatic effect for nearly 15 days, followed by a rapid bactericidal effect, thereby suggesting that ppk-1 is essential for M. tuberculosis growth in vitro and a potential drug target to combat tuberculosis (49). Rv3232c (ppk-2) has also been shown to be widely conserved in a number of human pathogens, regulates nucleotide diphosphate kinase activity, and is important for intracellular survival in mycobacteria (26). Rv0496 (ppx) has also been shown to possess polyphosphatase activity, showing a distinct preference for short-chain polyP as the substrate (50).…”

“…In bacteria, some of the enzymes involved in polyP metabolism are polyphosphate kinase 1 (PPK-1), which catalyzes the reversible transfer of the terminal (␥) phosphate of ATP to form polyP, and polyphosphatase (PPX), which processively hydrolyzes the terminal residues of polyP to liberate P i (21)(22)(23). Polyphosphate kinase 2 (PPK-2) is another enzyme involved in polyP metabolism that drives synthesis of GTP and ATP using polyP as a phosphate donor (24)(25)(26).…”

NUCLEOSIDE DIPHOSPHATE KINASE GENE IS EXPRESSED THROUGH MULTIPLE TRANSCRIPTS IN Mycobacterium smegmatis

“…The requirement for NTPs and dNTPs, during stress conditions, might be getting mediated through the marginal modulation of the levels of T2. Nevertheless, NDK is nonessential for most bacterial systems, including M. tuberculosis [20], as other enzymes such as adenylate kinase [52] and polyphosphate kinase [53,54], which are present in M. smegmatis also (TIGR Database), might be able to synthesise NTPs and dNTPs, as in E. coli [52,53]. Therefore, M. smegmatis may not be entirely dependent on NDK modulation for NTPs and dNTPs and this may probably be the reason for the lack of significant modulation of NDK.…”