Characterization of a thermostable and piezotolerant prolidase from the hyperthermophilic archaeon Pyrococcus yayanosii CH1

Abstract:

[Objective] Prolidase is an enzyme that can hydrolyze proline or hydroxyproline residues from the C-terminal dipeptides (Xaa-Pro). A putative prolidase-encoding gene was identified in the genome of Pyrococcus yayanosii CH1 isolated from the deep sea. In this study, we characterized the enzymatic properties of Pyprol encoded by PYCH_07700 in vitro, aiming to find a new prolidase. [Methods] Pyprol was heterologously expressed in the hyperthermophilic archaeon Thermococcus kodakarensis TS559. The dipeptide Met-Pro was used as a substrate to test the prolidase activity of the purified recombinant protein. [Results] Pyprol showed the best performance at 100 °C and pH 6.0. Pyprol binding to Co²⁺ exhibited the maximum activity, and the optimal metal ion concentration was 1.2 mmol/L. Pyprol had catalytic activity in a wider pH range and can tolerate higher concentrations of metal ions than the prolidase Pfprol from P. furiosus. Pyprol was a piezotolerant protein with an optimal hydrostatic pressure of 40 MPa. It exhibited enhanced activities at 40, 70, and 100 °C under 40 MPa, compared with at the atmospheric pressure. [Conclusion] Pyprol is a novel thermostable and piezotolerant prolidase of P. yayanosii CH1, which is an obligate piezophilic hyperthermophilic archaeon strain isolated from a deep-sea hydrothermal vent.