The structure of exopolyphosphatase (PPX) from Porphyromonas gingivalis in complex with substrate analogs and magnesium ions reveals the basis for polyphosphate processivity

[Display omitted] •Bacterial polyphosphatase (PPX) hydrolyzes polyP and plays vital role in stringent response.•Structure of PPX was solved in complexed with substrate analogs and active magnesium ions.•The PPX possesses a more closed conformation and hydrolyzed polyP more efficiently.•Arg255 penetrates into the cavity and acts as a gatekeeper of the substrate entry channel.•These results provides insights into the molecular mechanism and substrate specifity. The enzymes exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GppA) play important roles in the bacterial stringent response. PPX degrades inorganic polyphosphate (polyP), a polymer composed of a few to hundreds of phosphate residues supporting cell survival in the stationary phase. The crystal structure of PPX from Porphyromonas gingivalis (PgPPX) in complex with catalytic magnesium ions and several sulfate ions was solved. PgPPX contained two domains and represented a “closed” configuration. Four sulfate ions forming a linear dispersed chain were observed in the aqueduct of the PPX dimer, which the long polyP chain most likely occupied. The side chain of R255 stretched into the cavity where polyP could be located, obstructing the entrance of larger substrates such as NTP and NDP. This study provided the first view into the structure of the PPX/GppA homolog in complex with magnesium ions and substrate analogs and explained how PgPPX implemented its functionality.