Crystal structure of PqqC from Klebsiella pneumoniae at 2.1 Å resolution (2004)

The biosynthesis of pyrroloquinoline quinone (PQQ), a novel vitamin1 and redox cofactor of several quinoprotein dehydrogenases, is facilitated by an unknown pathway that requires the expression of six genes, pqqA-F.2 The final step is catalyzed by PqqC, which uses molecular oxygen to oxidize a quinone intermediate to PQQ.3 Here we report the purification and crystallization of PqqC and the determination of its X-ray crystal structure at 2.1 Å resolution. The pqqC open reading frame from Klebsiella pneumoniae NCBI accession no. X58778 (residues Met1 to VAL251) was amplified by PCR and cloned into expression plasmid pet21d (Invitrogen, Carlsbad). The protein was expressed in E.coli strain BL21(DE3). Cells (1 L) were induced with 0.5 mM IPTG (3 h at 37°C) harvested and frozen at −80°C. PqqC was purified by a combination of affinity and gel filtration chromatography. Briefly, after harvesting and lysis of the cells, the crude extract was centrifuged and applied to a 5 ml Ni-affinity column (Pharmacia). PqqC containing fractions were dialyzed against 20 mM Tris/HCl, at pH 8.0, 1 mM DTT, further purified on a Superdex 200 gelfiltration column (Pharmacia, Uppsala, Sweden), and concentrated to a final concentration of ∼8 mg/mL in an ultrafiltration cell (Amicon). The seleno-methionine substituted protein was expressed as described4 and purified accordingly to the wildtype with the exception that there was 5 mM DDT added to the dialysis and gelfiltration buffers. Crystals were grown at 25°C by the sitting drop vapor diffusion method in droplets composed of one part protein solution (8 mg/ml in 20 mM Tris/HCl, at pH 8.0 and 1mM DTT) and one part reservoir solution (1.2 M ammonium sulfate, MES/NaOH at pH 6.0). The orthorhombic crystals are of space group P21212 and contain two molecules in the asymmetric unit. For data collection, these crystals were transferred into cryobuffer (crystallization buffer with 25% (v/v) glycerol) and flash-cooled in liquid nitrogen. The three-wavelength Selenium MAD data set was collected from one single crystal, using synchrotron radiation at beamline 9.2 of SSRL. Oscillation data were recorded in frames of 1° through a continuous angular range of 120° for the peak (Se-λ1), the high energy remote (Se-λ2), and the inflection point (Se-λ3). Native datasets were collected at beamline 11.1 of SSRL. All data were processed with the programs DENZO and SCALEPACK.5 The PqqC structure was solved, phased and traced using programs SOLVE and RESOLVE.6 Crystallographic refinement was carried out using TLS refinement in REFMAC57 and program O.8 The PqqC model comprises two protein monomers (residues A1–A152, A160–A251, B1–B152, B162–B251) and 130 water molecules. No electron density was observed for residues A153–A159 and B153–B161. Crystallographic statistics are shown in Table I. The structure exhibits excellent stereochemistry with 94.1% and 5.9% of the residues in the most favored and additional allowed regions, respectively, of the Ramachandran plot.9 Surface calculations were carried out with the CASTP-server10 and the protein-protein interaction server.11 The figure was drawn with PYMOL (DeLano Scientific LLC). Coordinates and structure factors for PqqC have been deposited with the Protein Data Bank (http://www.rcsb.org/pdb) under accession code 1OTV. The PqqC protein from Klebsiella pneumoniae has a molecular weight of 28,914 Da (251 residues) and a calculated isoelectric point of 5.6. PqqC was cloned, over-expressed in E. coli, purified and crystallized. PqqC crystallizes in the orthorhombic space group P21212 with unit cell dimensions a = 67.15 Å, b = 117.03 Å, c = 74.96 Å, and two molecules in the asymmetric unit. The structure of the enzyme was determined by a MAD experiment using a seleno-methionine substituted protein.4 The PqqC model comprises two protein monomers (residues A1–A152, A160–A251, B1–B152, B162– B251) and 130 water molecules. Statistics for data collection, refinement and model quality are summarized in Table I. The PqqC molecule is roughly cylindrical, with approximate dimensions of 45 Å × 29 Å × 37 Å [Fig. 1(A)]. PqqC folds into a compact seven helix bundle that resembles the heme-oxygenase fold,14 with six circular aligned helices (H1, H2, H3, H4, H5, H7) partly embracing a seventh hydrophobic helix (H6). Helices H1–H7 form a long up and down helix bundle, where helices H7, H1, H5, H2 and H4 pack parallel and helices H5, H6, H3, H2, H4 and H7 pack anti-parallel to each other. Helices H1, H3, H4, H5, H6 are kinked and can therefore be represented as separate shorter helices denoted as A and B. This is especially true for helix H3, where the peculiar hairpin loop L3, residues 84 to 92, is inserted [Fig. 1(A)]. Crystal structure of PqqC. A: The PqqC dimer depicted in ribbon representation rainbow color coded by residue numbering from N-terminus (blue) to C-terminus (red), with Helices H1-H7 and loop L3 labeled. B: The active site cavity of PqqC. A cut through the PqqC monomer is shown in surface representation color coded according to surface potential (blue positive; red negative). For size comparison a manually docked PQQ molecule is depicted in sticks. The active site carries a significant positive charge. The two channels connecting the active site to the solvent are marked with arrows E1 and E2. C: The PqqC dimer shown in surface representation color coded according to surface potential. A series of views with consecutive 90° rotations are displayed. PqqC forms a homo-dimer in solution, as evident from gel filtration experiments (data not shown). The dimer is formed through an interaction via helices H1, H4b, H7 and the C-terminus with a buried surface area of 2145 Å2 per monomer [Fig. 1(A,C)]. The nature of the interaction is mainly hydrophobic and facilitated by 63% non-polar atoms. The polar interface residues H26, R206, E209, D216, Y236, T238, H246, T247, T248, L250, and R233 contribute 19 hydrogen bonds.10 Analysis of the PqqC structure shows that the seven α-helices provide the scaffold for a unique active site cavity [Fig. 1(B)]. The cavity is lined with 42 mostly hydrophilic and aromatic residues that are highly conserved within PqqC proteins from different bacteria. Interestingly, these residues are contributed from all seven helices H1 to H7. The cavity shows a distinct overall positive charge, measures 9 Å × 13 Å × 23 Å and embraces a molecular surface volume of 2200 Å3.11 Two openings connect it to the outside. The larger opening (E1) between helices H1 and H6 is divided by the disordered region between helices H5a and H5b and extends over a molecular surface area of 200 Å2. The mouth region is lined by 14 residues, including the histidine ladder H24, H30 and H34. The second opening (E2) is much smaller and has a molecular surface of 30 Å2. It penetrates the surface of the protein between helices H6a, H6b and the unique loop L3. The opening is guided by residues R179, D83, Q182 and N184. Given the size of the two openings, the transfer of substrate and product is only possible via the large entrance. To our surprise the PqqC structure does not appear to contain a metal-site or a redox-cofactor binding site as would be expected for an oxidase, raising the question of how the final oxidative step of PQQ-biosynthesis is achieved. A structural similarity search performed with the atomic coordinates of PqqC using the DALI server15 yields human heme-oxygenase,14 with an RMSD of 3.1 Å for the superposition of 194 Cα atoms and 9% sequence identity, the R2 subunit of ribonucleotide reductase,16 with an RMSD of 3.7Å for the superposition of 172 Cα atoms and 15% sequence identity, and methane monooxygenase,17 with an RMSD of 3.9 Å for the superposition of 170 residues and 12% sequence identity. Intriguingly, despite the fact that none of the active site residues are conserved, each of them appears to be a redox enzyme, suggesting that this fold may be particularly suitable for this type of enzyme. RS was supported by FWF-fellowship J2209-B04. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a National user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences.