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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.

Characterization of the PH1704 Protease from Pyrococcus horikoshii OT3 and the Critical Functions of Tyr120