Figure 1.
Sequence alignment and phylogenetic tree of PH1704.
A, Sequence alignment of PH1704 with other members in the DJ-1 superfamily. PfpI (gi/18978091) 90% sequence identify with PH1704; DR1199 (PDB Id 2VRN), 46% sequence identify with PH1704; ThiJ from E. coli (gi/190906193), 27% sequence identify with PH1704; YDR533Cp (PDB Id 1RW7) 25% sequence identify with PH1704; Human DJ-1 (PDB Id 1J42), 24% sequence identify with PH1704; Hsp31 (PDB Id 1N57) 19% sequence identify with PH1704. The color purple represent for similar sequence, and color green represent for identical sequence. B, a phylogenetic tree of the DJ-1/ThiJ/PfpI superfamily and several closely related sequences. The tree contains three more closely related clades: Hsp31 is in the Hsp31 family class I, whereas PH1704 and PfpI are in the Hsp31 family class III, with the homolog of the same family.
Figure 2.
The active pocket compare with PH1704 (green) and Hsp31 (Gray).
The color orange dot represent for the active pocket for PH1704 (the active pocket size of PH1704 is 1622.1 Å3, calculated by CASTp server [37] with 1.4 Å), color red dot represent for the active pocket for Hsp31 (the active pocket size of is 980 Å3, calculated by CASTp server with 1.4 Å). It can be concluded that the active pocket size of PH1704 is larger than that of Hsp31.
Figure 3.
Cloning, expression, validation and proteolytic activity of PH1704.
A, Lane 1: Hind III Marker; lane 2: Pyrococcus horikoshii genome; Lane 3: DL2000 Marker; lane 4: PCR product using P. horikoshii genome as template. B, Lane 1: High molecular weight protein marker; lane 2: PH1704 crude extract after heated at 80 oC for 15 min; lane 3: The purified recombinant PH1704 protease on non-denaturing gel; lane 4: Western blotting analysis of the purified PH1704; lane 5: enzyme assays of proteolytic activity on 10% Non-denaturing PAGE with 0.1% gelatin;lane 6: negative control on 10% Non-denaturing PAGE with only 0.1% gelatin substrate.
Table 1.
Purification of the recombinant PH1704 from Pyrococcus horikoshi OT3.
Figure 4.
Effects of pH and temperature on the recombinant PH1704's activity.
A, The temperature activity profile; B, pH dependence reactions catalyzed by PH1704; Substrate specificity of recombinant PH1704. The aminopeptidase activity towards L-R-amc was defined as 100%; C, The aminopeptidase substrate specificity; D, The endopeptidase substrate specificity.
Table 2.
Effects of metal ions on enzyme activity (%).
Table 3.
Effects of inhibitors on enzyme activity (%).
Figure 5.
All 29 docked ligands located in AC contacts.
Figure 6.
The active residues (Glu12, Glu15, Lys43, Gly70, Arg71, Cys100, His101, Tyr120, Val150, Arg471, Glu474, and Arg475) around the substrate, R-amc and AAFR-amc.
Figure 7.
The mechanism for PH1704 is explained by the activated nucleophile (Cys100) attacking the hydroxyl carbon of the substrate to form a tetrahedral intermediate.
His101 and Glu474 function as catalytic base.
Table 4.
Averaged structural interaction fingerprints calculated over all successfully docked poses among two receptor conformations and 29 ligands presented for all identified interacting residues.
Table 5.
Kinetic parameters for hydrolytic substrates of l-R-amc and L-AAFR-amc.
Figure 8.
The dual-function of Tyr120: participation in enzyme nucleophilic catalytic through a hydrogen bond; acting as entrance gate with Lys43.
A, hydrogen bond between Cys100 and Tyr120; B, 29 substrates docked in AC contacts; C, The entrance gate guide by Tyr120 and Lys43 (R-amc in the active sit).