Abstract
Inorganic polyphosphate (poly-P), guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) are ubiquitous in bacteria. These molecules play a variety of important physiological roles associated with stress resistance, persistence, and virulence. In the bacterial pathogen Mycobacterium tuberculosis, the identities of the proteins responsible for the metabolism of polyphosphate and (p)ppGpp remain to be fully established. M. tuberculosis encodes two PPX-GppA homologues, Rv0496 (MTB-PPX1) and Rv1026, which share significant sequence similarity with bacterial exopolyphosphatase (PPX) and guanosine pentaphosphate 5′-phosphohydrolase (GPP) proteins. Here we delineate the respective biochemical activities of the Rv0496 and Rv1026 proteins and benchmark these against the activities of the PPX and GPP proteins from Escherichia coli. We demonstrate that Rv0496 functions as an exopolyphosphatase, showing a distinct preference for relatively short-chain poly-P substrates. In contrast, Rv1026 has no detectable exopolyphosphatase activities. Analogous to the E. coli PPX and GPP enzymes, the exopolyphosphatase activities of Rv0496 are inhibited by pppGpp and, to a lesser extent, by ppGpp alarmones, which are produced during the bacterial stringent response. However, neither Rv0496 nor Rv1026 have the ability to hydrolyze pppGpp to ppGpp; a reaction catalyzed by E. coli PPX and GPP. Both the Rv0496 and Rv1026 proteins have modest ATPase and to a lesser extent ADPase activities. pppGpp alarmones inhibit the ATPase activities of Rv1026 and, to a lesser extent, the ATPase activities of Rv0496. We conclude that PPX-GppA family proteins may not possess all the catalytic activities implied by their name and may play distinct biochemical roles involved in polyphosphate and (p)ppGpp metabolic pathways.
Highlights
All living cells appear to have the physiological ability to synthesize and degrade inorganic polyphosphate molecules
The dimeric arrangements of the E. coli polyphosphate glucokinase (GK); and exopolyphosphatase (PPX) and guanosine pentaphosphate 59-phosphohydrolase (GPP) proteins established here is in agreement with previous reports [12,16], as well as the X-ray crystal structures previously obtained for the E. coli PPX protein [14,15]
The sequences of the MTB-PPX1, Rv1026 and CG-PPX2 proteins align with the N-terminal regions of the E. coli PPX and GPP
Summary
All living cells appear to have the physiological ability to synthesize and degrade inorganic polyphosphate (poly-P) molecules. These linear biopolymers comprise chains of phosphate (Pi) residues linked via ‘high-energy’ phosphoanhydride bonds, and range from a few to several hundred phosphate residues in length. Poly-P is involved in a diverse range of biochemical, physicochemical and biological processes; e.g. the modulation of membrane structure and permeability; cell morphogenesis; DNA replication; as well as RNA and protein degradation. Bacterial polyphosphate metabolism is of notable environmental importance, playing a key role in the biological removal of phosphate from wastewater [11]. The modulation of intracellular poly-P concentrations is of pivotal importance to numerous physiological processes involved in bacterial growth, viability, adaptability and infection
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