title: Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.
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 Kcat and Kcat/Km 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, Kcat and Kcat/Km 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.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Science, Jilin University, Changchun, China; College of Food Science and Engineering, Jilin Agricultural University, Changchun, China.
authors.forename: Dongling
authors.initials: D
authors.lastname: Zhan
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Science, Jilin University, Changchun, China.
authors.forename: Aixi
authors.initials: A
authors.lastname: Bai
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Science, Jilin University, Changchun, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Science, Jilin University, Changchun, China.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Han
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Science, Jilin University, Changchun, China; State Key Laboratory of Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D010450:Endopeptidases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008024:Ligands; D008670:Metals; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D059748:Proteolysis; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D007477:Ions; D008024:Ligands; D008670:Metals; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D010450:Endopeptidases