Crystal Structural and Functional Analysis of the Putative Dipeptidase from Pyrococcus horikoshii OT3

Abstract

The crystal structure of a putative dipeptidase (Phdpd) from Pyrococcus horikoshii OT3 was solved using X-ray data at 2.4 A resolution. The protein is folded into two distinct entities. The N-terminal domain consists of the general topology of the alpha/beta fold, and the C-terminal domain consists of five long mixed strands, four helices, and two 3(10) helices. The structure of Phdpd is quite similar to reported structures of prolidases from P. furiosus (Zn-Pfprol) and P. horikoshii (Zn-Phdpd), where Zn ions are observed in the active site resulting in an inactive form. However, Phdpd did not contain metals in the crystal structure and showed prolidase activity in the absence of additional Co ions, whereas the specific activities increased by 5 times in the presence of a sufficient concentration (1.2 mM) of Co ions. The substrate specificities (X-Pro) of Phdpd were broad compared with those of Zn-Phdpd in the presence of Co ions, whose relative activities are 10% or less for substrates other than Met-Pro, which is the most favorable substrate. The binding constants of Zn-Phdpd with three metals (Zn, Co, and Mn) were higher than those of Phdpd and that with Zn was higher by greater than 2 orders, which were determined by DSC experiments. From the structural comparison of both forms and the above experimental results, it could be elucidated why the protein with Zn(2+) ions is inactive.

Figure 1

(a) Ribbon diagram of Phdpd (monomer form) showing N and C-terminal domains. α-helix, 3₁₀-helices, β-strands, and loops colored magenta, red, cyan, and yellow, respectively. The figure was made by the programs MOLSCRIPT and Raster3D [20, 21]. (b) Dimer form of Phdpd. (c) Electrostatic potential surface of the Phdpd and its active site pocket. The positively and the negatively charged surface regions are noted in blue and red, respectively.

Figure 2

DSC curves of Phdpd and Zn-Phdpd in the presence of metal ions. (a) and (b) represent the DSC curves of Phdpd and Zn-Phdpd, respectively. All protein concentrations were 0.01 mM. DSC curves 1, 2, 3, and 4 were measured in the presence of 0 M metal, 0.02 mM Zn, 0.02 mM Co, and 0.02 mM Mn, respectively, in 50 mM Tris buffer at pH 7.8. The scan rate was 100 K h⁻¹.

Figure 3

DSC curves of Phdpd and Zn-Phdpd in various concentrations of Co ion. (a) and (b) represent the DSC curves of Phdpd and Zn-Phdpd, respectively. All protein concentrations were 0.01 mM. DSC curves 1, 2, 3, and 4 were measured in the presence of 0, 0.01, 0.02, and 0.05 mM Co, respectively, in 50 mM Tris buffer at pH 7.8. The scan rate was 100 Kh⁻¹.

Figure 4

(a) Stereoview of the superposition of Phdpd (Cyan) with Zn-Phdpd (Yellow) and Zn-Pfprol (Pink) structures. (b) Stereoview of the conserved active site residues superimposed between the Phdpd (cyan) and Zn-Phdpd (yellow). Two active site metal atoms of Zn-Phdpd and two water molecules (W278 and W279) of Phdpd are shown in yellow and red color spheres, respectively. The side chains of the conserved active site residues are labeled according to Phdpd sequence numbering.

Figure 5

Structure-based sequence alignment of dipeptidase (PDB ID: 2HOW from P. horikoshii OT3 (Phdpd) with prolidase from P. furiosus (Zn-Pfprol) and dipeptidase (PDB ID: 1WY2) from P. horikoshii OT3 (Zn-Phdpd). Above and below the alignment are shown the secondary structure elements. Alignment was performed using CLUSTAL W [37], and the figure was produced using ESPript [38].

Figure 6

Deviations of Cα atoms after the Cα superposition between Phdpd and Zn-Phdpd. Downward and upward arrows represent the positions of H198, D215, D226, H290, E319, and E333 which are important residues in the active site.

Figure 7

Stereoview of selected active site pocket residues of Phdpd (cyan) superimposed on Zn-Phdpd (yellow). Active site pocket residues are shown by the ball and stick model and the metal ions are shown as spheres. His195 of Zn-Phdpd corresponds to His198 of Phdpd.