Abstract
The PH1704 protease from hyperthermophilic archaean Pyrococcus horikoshii OT3 is a member of DJ-1/ThiJ/PfpI superfamily with diverse functional subclasses. The recombinant PH1704 was efficiently purified and was systematically characterized by a combination of substrate specificity analysis, steady-state kinetics study and molecular docking research. The homogeneous protease was obtained as a presumed dodecamer with molecular weight of ∼240 kDa. Iodoacetamide strongly inhibited the peptidase activity, confirming that Cys100 is a nucleophilic residue. The recombinant protein was identified as both an aminopeptidase and an endopeptidase. Experimental data showed that L-R-amc was the best substrate of PH1704. Structural interaction fingerprint analysis (SIFt) indicated the binding pose of PH1704 and showed that Tyr120 is important in substrate binding. Kinetic parameters K cat and K cat /K m of the Y120P mutant with L-R-amc was about 7 and 7.8 times higher than that of the wild type (WT). For the endopeptidase Y120P with AAFR-amc, K cat and K cat /K m is 10- and 21- fold higher than that of WT. Experimental data indicate the important functions of Tyr120: involvement in enzyme activity to form a hydrogen bond with Cys100 and as an entrance gate of the substrate with Lys43. The results of this study can be used to investigate the DJ-1/ThiJ/PfpI superfamily.
Highlights
Pyrococcus horikoshii is a thermophilic archaean with an optimal growth temperature about100uC and grows at a greater sea depth than the other archaea [1]
The PH1704 protease from Pyrococcus horikoshii OT3 is a hyperthermophilic enzyme that belongs to the DJ-1/ ThiJ/Pyrococcus furiosus protease I (PfpI) superfamily [2]
Heat-shock protein 31(Hsp31) was characterized as a chaperone and a peptidase [5,6,7], Pyrococcus furiosus protease I (PfpI) exhibited protease/peptidase activity [8,9], PH1704 is regarded as a protease [2,10], and Escherichia coli YhbO is involved in the response to hyperosmotic or acid stress [11]
Summary
Pyrococcus horikoshii is a thermophilic archaean with an optimal growth temperature about100uC and grows at a greater sea depth than the other archaea [1]. The PH1704 protease from Pyrococcus horikoshii OT3 is a hyperthermophilic enzyme that belongs to the DJ-1/ ThiJ/PfpI superfamily [2]. An increasing number of studies on this superfamily are being conducted. Despite this growing interest, few members of this superfamily have been biochemically characterized. Heat-shock protein 31(Hsp31) was characterized as a chaperone and a peptidase [5,6,7], Pyrococcus furiosus protease I (PfpI) exhibited protease/peptidase activity [8,9], PH1704 is regarded as a protease [2,10], and Escherichia coli YhbO is involved in the response to hyperosmotic or acid stress [11]
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