Crystal structure of acylphosphatase from hyperthermophilic archaeon Pyrococcus horikoshii OT3

Analysis of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 genome database led to the discovery and cloning of acylphosphatase (ORF PH0305a). To elucidate the first structure of archaeal acylphosphatase, we determined the crystal structure of P. horikoshii acylphosphatase at 1.72 Å resolution. The space group of the crystals was P3221, with unit-cell parameters a = b = 86.6 Å and c = 75.4 Å. The overall fold of P. horikoshii acylphosphatase was very similar to the structures of the eukaryotic enzymes. The conformation of putative active site was highly conserved.

revealed that two conserved residues, Arg23 and Asn41, are important for enzyme activity. Arg23 is involved in the binding of the phosphate moiety of the substrate, and Asn41 has been recognized as the catalytic residue involved in the orientation and stabilization of catalytic water molecule. 8) Pyrococcus horikoshii OT3 is a hyperthermophilic archaeon that was isolated from a hydrothermal fluid. P. horikoshii OT3 genome data indicate that this hyperthermophilic archaeon has the AcP gene (ORF PH0305a). 10) The protein encoded by this ORF consists of 91 amino acid residues with a molecular weight of 10260.
To elucidate the first structure of archaeal AcP, we crystallized P. horikoshii AcP and determined its three dimensional structure by X-ray crystallography. The overall fold of P. horikoshii AcP was very similar to the structures of eukaryotic enzymes, except for the loop structure near the C-terminus. The structure of putative active site was highly conserved.
Materials and methods. P. horikoshii AcP was overexpressed in E. coli, purified, and crystallized as described. 11) Protein samples were concentrated to 10 mg/ml for crystallization. All crystallization experiments were performed using the sitting-drop vapor-diffusion method at 293 K, and 1 µl of protein solution was mixed with 1 µl of reservoir solution. The best crystals were obtained after 2 days using the following reservoir composition: 0.7-0.9 M K/Na tartrate and 100 mM citrate buffer (pH 5.5).
Crystals were transferred into a cryo-protectant solution containing 0.8 M K/Na tartrate, 100 mM citrate buffer (pH 5.5), and 20% ethylene glycol before being picked up and flash-cooled in a nitrogen stream. Diffraction data were collected at BL41XU in SPring-8 at 100 K using a MAR CCD detector system to a resolution of 1.72 Å. Data were processed with DENZO/SCALEPACK.

12)
The crystals belonged to hexagonal space group P3 1 21 or , respectively). 13) The structure of the P. horikoshii AcP was determined by the molecular replacement method. Molecular replacement was performed with the program MOLREP from the CCP4 suites 14) using the coordinates of bovine CT-AcP (PDB code 2ACY 8) ). MOLREP was run using the data with a resolution range of 30-3 Å in both space groups P3 1 21 and P3 2 21. The best solution was obtained when we searched two monomers in the asymmetric unit in space group P3 2 21. That solution had an initial correlation coefficient of 0.334 and an R factor of 53.7%. Five percent of the reflections were excluded from the total for cross-validation with the R free . Initial structural refinements were carried out with CNS 15) using diffraction data to 1.72 Å with several cycles of torsion-angle simulated annealing with an initial temperature of 2500 K, energy minimization, and individual Bfactor refinement. After the structural refinements using CNS, structural refinements and auto model building were performed using ARP/wARP. 16) After auto model building, several cycles of manual model rebuilding and model refinement were performed using XtalView 17) and Refmac5. 18) Water molecules were picked up from an F o -F c map on the basis of peak heights and distance criteria. In the course of the water picking, four unexplained high electron density peaks were found. These were assigned as chloride ions and potassium ions considering the crystallization condition, the location where peaks observed, and the peak height of a 2 F o -F c and an F o -F c map. Chloride was contained in the protein solution for crystallization as sodium chloride, and potassium was contained in the reservoir solution as sodium potassium tartrate. Evaluation of the quality of the model was performed with PROCHECK. 19) The coordinates have been deposited into the Protein Data Bank with the accession number 1V3Z. Results and discussion. The crystal structure of P. horikoshii AcP was solved by molecular replacement at 1.72 Å resolution and refined to an R factor of 16.9% and an R free of 19.0% with a good geometry. The asymmetric unit contained two molecules of P. horikoshii AcP. The final electron density allowed positioning of 90 residues in each molecule. We could not determine the position of the N-terminal methionine residue. The final model contained two chloride ions, two potassium ions, and 178 ordered water molecules. In the Ramachandran plot, 20) 95.2% of the residues fell within the most favored regions, and the rest fell within the additionally allowed regions.
The structure of P. horikoshii AcP was superposed with the known structures of other AcPs (bovine CT-AcP: PDB code 2ACY 8) and AcPDro2: PDB code 1URR 9) ). This revealed that the structure of P. horikoshii AcP is very similar to those of other AcPs, except for a long loop positioned between β4 and β5. The root mean square deviation (r.m.s.d.) values calculated for the C α atoms were 0.66 Å (superposition of 84 residues of P. horikoshii AcP with bovine CT-AcP) and 0.88 Å (superposition of 82 residues of P. horikoshii AcP with AcPDro2).
Comparison of the structure and sequence of P. horikoshii AcP with those of eukaryotic AcPs allows us to identify the enzyme active site. The sequence stretch Gln15 -Arg20 forms a cradle-like conformation close to the N-terminal of the α1 helix (Fig. 2(A)). In this region, the nitrogen atoms of the main chain point toward the center of the cradle, where the phosphate moiety of the substrate is expected to bind. Amino acid residues in this region are highly conserved among bovine CT-AcP, AcPDro2, and P. horikoshii AcP. This phosphate recognition mechanism is adapted by the lowmolecular-weight phosphotyrosine protein phosphatases (LMW-PTPs). 21)- 23) The critical residues for AcP enzyme activity have been suggested previously. In bovine CT-AcP, Arg23 and Asn41 are indispensable as the main phosphate binding residue and as the residue involved in the orientation and stabilization of catalytic water molecule, respectively. 8) These residues are conserved as Arg20 and Asn38 in  P. horikoshii AcP. In the crystal structure of P. horikoshii AcP, we found chloride ion in the active site ( Fig. 2(A)). Chloride ion is located at the center of the cradle-like pocket where sulfate and chloride ions, competitive inhibitors for AcPs, are positioned in the bovine CT-AcP structure. The chloride ion found in P. horikoshii AcP would also inhibit enzyme activity. The conformation of active site was highly conserved among the CT-AcP, AcPDro2, and P. horikoshii AcP. The r.m.s.d. value calculated for the main chain atoms involved in the active site (Glu15 -Arg20 and Asn38) were 0.20 Å (superposition of 28 atoms of P. horikoshii AcP with bovine CT-AcP) and 0.27 Å (superposition of 28 atoms of P. horikoshii AcP with AcPDro2) (Fig. 2(B)).