A unique catalytic triad revealed by the crystal structure of APE0912, a short‐chain dehydrogenase/reductase family protein from Aeropyrum pernix K1 (2008)

Short-chain dehydrogenases/reductases (SDRs), about 250-residue long, are enzymes that catalyze NAD(P)(H)-dependent oxidation/reduction of various substrates such as alcohols, sugars, steroids, aromatic compounds, and xenobiotics.1-3 SDR family proteins have the N-terminal Rossman fold made up of 7 to 8 β-strands, which binds the cofactor NAD(P)(H), and a catalytic triad composed of Ser-Tyr-Lys.4 The APE0912 gene of hyperthermophilic archaea Aeropyrum pernix K1 encodes an SDR family protein with an unknown function.5 We have determined the crystal structure of APE0912 at 1.8-Å resolution. The overall structure of APE0912 is similar to those of other SDR family members, but APE0912 does not have a catalytic triad Ser-Tyr-Lys characteristic of the SDR family members. Instead, APE0912 has a unique Ser-Ser-Arg triad. The two molecules in the asymmetric unit exhibit open and closed forms that alter the accessibility of substrates and cofactors to the Ser-Ser-Arg triad. Here, we report the crystal structure of APE0912 with its unique catalytic triad and the structural difference between open and closed forms. The gene of APE0912 from A. pernix K1 was amplified by PCR and cloned into the pET-26b(+) expression vector (Novagen). The protein without any tags was overexpressed in E. coli Rosetta (DE3) (Novagen). Harvested cells were resuspended in 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl, and then disrupted by sonication. The lysate was centrifuged at 40,000g at 4°C for 30 min. The supernatant was incubated at 80°C for 30 min, and then centrifuged at 40,000g at 4°C for 30 min. The thermostable fraction was supplemented with a final concentration of 0.2% polyethyleneimine, and centrifuged at 40,000g at 4°C for 30 min to remove any nucleic acids. Ammonium sulfate was added to the supernatant to 70% saturation at 25°C, and then centrifuged at 40,000g at 4°C for 30 min. The precipitates were solubilized into 50 mM MES-NaOH (pH 6.0), 200 mM NaCl, and 10 mM NADPH. The solution was loaded onto a Resource S (GE Healthcare Bioscience) column equilibrated with 50 mM sodium phosphate buffer (pH 6.0), 200 mM NaCl, and 10 mM NADPH, and eluted with a linear gradient of 200–700 mM NaCl. The purified APE0912 was dialyzed against 50 mM MES-NaOH (pH 6.0), 200 mM NaCl, and 5 mM NADPH, and concentrated to a protein concentration of 10 mg/mL. The sitting drop vapor diffusion method was used for crystallization. One microliter each of purified APE0912 solution (10 mg/mL) and the reservoir solution were mixed to prepare a crystallization drop, and the drop was equilibrated against 200 μL of the reservoir solution. Crystals of APE0912 were grown at 293 K with the reservoir solution, 100 mM Tris-HCl (pH 8.4), and 5% PEG 8000 (Hampton Research). The crystals were picked up with mounting loops and frozen directly in liquid nitrogen using 20% ethylene glycol as a cryoprotectant. X-ray diffraction data of the APE0912 crystal were obtained on the KEK beamline NW12 (Tsukuba, Japan) to 1.8-Å resolution. The crystal belonged to the space group C2, with unit cell dimensions a = 110.76 Å, b = 68.87 Å, c = 69.34 Å, and β = 119.0°. The X-ray diffraction data were processed with the program package HKL20006 and the CCP4 suite.7 The initial structure was obtained by the molecular replacement method using the program Molrep8 in the CCP4 suite. The search model used was the crystal structure of TM1169 (PDB code: 1O5I). The initial structure was refined using Xfit,9 Coot,10 and Refmac511 in the CCP4 suite. The stereochemistry of the structure was checked by the program PROCHECK.12 The purified APE0912 was loaded onto a Superdex 200 HR 10/30 (Amersham Biosciences) column equilibrated with 50 mM sodium phosphate buffer (pH 7.0) and 150 mM NaCl, and eluted with the same buffer at a flow rate of 0.5 mL/min. The molecular weight of APE0912 was calculated by comparing its elution volume with that of appropriate standard proteins. The crystal structure of APE0912 was determined at 1.8-Å resolution by molecular replacement. The final structure includes a homodimer (residues 1–196, 220–260 for chain A, and residues 1–198, 216–260 for chain B), 361 water molecules, and two Na+ ions in the asymmetric unit. No electron density was observed for the remaining residues. The Matthews coefficient (Vm)13 was 2.07 Å3/Da, and the estimated solvent content was 40.7%. The Ramachandran plot showed that 94.2% of the residues are in most favored regions, and all the residues are within the additional allowed regions. The data collection and refinement statistics are summarized in Table I. The APE0912 protomer contains seven β-strands, nine α-helices, and four 310-helices (Fig. 1). The gel filtration chromatogram of APE0912 shows that it forms a dimer and tetramer in solution, with a peak volume ratio of 10:1. The quaternary structure prediction using PISA server15 suggests two possible modes of dimeric assembly with buried areas of 3424 Å2 and 3339 Å2, 16% of the surface area of each protomer [Fig. 2(A)], and a possible mode of tetrameric assembly with a buried area of 13560 Å2, 32% of the surface area of each protomer, in the crystal [Fig. 2(B)]. The dimeric assembly depicted to the left in Figure 2(A) is normally seen in homodimeric SDRs while that depicted to the right in Figure 2(A) is only observed in 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSDH/CR) of Comamonas testosteroni (PDB codes: 1FJH and 1FK8).16 The tetrameric assembly is obtained by expanding either of the dimeric assemblies by the crystallographic twofold axis. APE0912 belongs to the superfamily of NAD(P)-binding Rossmann-fold domains and the family of tyrosine-dependent oxidoreductases (also known as the SDR family) in SCOP (Structural Classification of Proteins).17 No electron density for NADPH was observed, although APE0912 has an NAD(P)-binding Rossmann-fold composed of a parallel β-sheet sandwiched between two arrays of parallel α-helices,18 and the protein solution containing 5 mM NADPH was used for crystallization. All members of the SDR family reported to date have a catalytic triad of Ser-Tyr-Lys. But the sequence alignment with ClustalW19 indicates that APE0912 does not have the catalytic triad residues characteristic of the SDR family. Instead, APE0912 has Ser144, Ser157, and Arg161 at the corresponding positions (Fig. 3). Overall structure of APE0912 protomer (chain A). The N- and C- termini are labeled as N and C. β-strands (β1–7), α-helices (α1–9), and 310-helices (η1–4) are shown in yellow, red, and blue, respectively. The Ser-Ser-Arg triad is shown with a stick diagram and labeled as S144, S157, and R161. Residues 197–219 are missing in the structure (shown by the dotted line). This figure was drawn with PyMOL.14 (A) Two possible dimeric assemblies of APE0912 with a buried area of 3424 Å2 (left) and 3339 Å2 (right). (B) Tetrameric assembly of APE0912. In all figures, chains A and B are shown in yellow and blue, respectively. This figure was drawn with PyMOL.14 Sequence conservation among the SDR family proteins. PDB code 1YBV: 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea, 2HSD: 3α,20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans, 1CYD: Mouse lung carbonyl reductase, 1FDU: Human 17β-hydroxysteroid dehydrogenase. As shown by the blue arrowheads, the catalytic triad is not conserved in APE0912. The secondary structure elements of APE0912 chains A and B are shown above the alignment. The positional alteration of α8 is indicated by the red arrows. This figure was prepared by ESPript.20 The two molecules of APE0912 in the asymmetric unit are not identical. The structural difference is seen in the substrate binding loop, residues 189–226, where residues 197–219 for chain A and 199–215 for chain B are not modeled due to the lack of electron densities. This loop region forms a roof above the Ser-Ser-Arg triad and can control the accessibility of the substrates and the cofactor to the triad.16 Chains A and B correspond to the closed and open forms, respectively (Fig. 4). When chains A and B are superimposed by matching Cα atoms of the Ser-Ser-Arg triad, the distance between the Cα atoms of Arg196 in chains A and B is 11.4 Å, while the overall r.m.s. deviation between the Cα atoms in chains A and B is 1.9 Å. Interestingly, α8 of chain A at residues 188–193 has gone slack in chain B, and another helix appears at residues 192–198 [Figs. 3 and 4(C)]. This positional alteration of α8 between chains A and B may be important for the conformational changes between the open and closed forms of APE0912. Although both the open and closed forms of SDR family protein have already been reported,21, 22 the positional alteration of the helix α8 found in APE0912 is unique among other SDR family members. (A) Surface diagram of APE0912 chain A. Residues 189–196 are shown in blue. (B) Surface diagram of APE0912 chain B. Residues 189–198 are shown in green. The Ser-Ser-Arg triad (shown in red) can only be seen in chain B (open form). (C) Superposed diagram of chains A (shown in light blue) and B (shown in light green). There is a structural alteration between chain A residues 189–196 (shown in blue) and chain B residues 189–198 (shown in green). Note that all diagrams are presenting the same side of the molecule. This figure was drawn with PyMOL.14 A structural similarity search performed with the atomic coordinates of APE0912 using DALI server23 has revealed that APE0912 is most similar to 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea (PDB code: 1YBV).24 The r.m.s. deviation between 212 pairs of Cα atoms of APE0912 and 1YBV is 1.4 Å. 1YBV has the typical catalytic triad of Ser164, Tyr178, and Lys182, with a similar spatial distribution to the unique Ser-Ser-Arg triad of APE0912 (Fig. 5). Although the activity of APE0912 has not yet been specified, the Ser-Ser-Arg triad in APE0912 seems to act in the same way as the Ser-Tyr-Lys catalytic triad of other SDRs. The distances between Nη1 of Arg161 and Oγ of Ser144 or Ser157 in APE0912 are shorter than the distances between Nζ of Lys182 and Oγ of Ser164 or Oη of Tyr178 in 1YBV due to the lack of cofactor NADPH in APE0912. (A) The putative catalytic triad Ser-Ser-Arg of APE0912. (B) The catalytic triad Ser-Tyr-Lys of 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea. The spatial distribution of the triad is similar between the two structures. This figure was drawn with PyMOL.14 The atomic coordinates and structure factors have been deposited in the Protein Data Bank with the PDB code of 2Z1N. The synchrotron-radiation experiments were performed at KEK beamline NW12 (Tsukuba, Japan) with the approval of the Japan Synchrotron Radiation Research Institute (Proposal No. 2003S2-002).