Crystal structure of an alanine‐glyoxylate aminotransferase from <i>Anabaena</i> sp. at 1.70 Å resolution reveals a noncovalently linked PLP cofactor (2005)

In order to extend the structural coverage of the eukaryotic proteins in the Protein Family database (Pfam),1 we selected 331 bacterial homologues of proteins represented in the mouse genome from the American Type Culture Collection (ATCC) proteome database (http://www.ebi.ac.uk/integr8). One of these homologues, the alanine-glyoxylate aminotransferase (AGT) from the cyanobacterium Anabaena [Nostoc.] sp. PCC 7120 (GI: 17130350) belongs to subclass-V of aminotransferases [Enzyme Commission (EC): 2.6.1.44].2 AGT is a pyridoxal-phosphate enzyme involved in serine–glycine metabolism, where it catalyses the transamination of L-alanine and glyoxylate to pyruvate and glycine. Human AGT shares 42% sequence identity with the bacterial AGT, but functions as a bifunctional enzyme with an additional serine-pyruvate transaminase (SPT) activity (EC 2.6.1.51).3 In humans, a deficiency of liver-specific peroxisomal AGT causes primary hyperoxaluria type 1 (PH1), which is responsible for the potentially lethal, hereditary kidney stone disease.4 Anabaena sp. AGT has a molecular weight of 41,736 Da (residues 1–381) and a calculated isoelectric point of 5.8. Here, we report the crystal structure of AGT determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).5 The crystal structure of Anabaena sp. AGT 17130350 [Fig. 1(A)] was determined to 1.70 Å resolution using the single-wavelength anomalous dispersion (SAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes 1 protein monomer (residues 5–381), 1 pyridoxal-5′-phosphate (PLP), and 423 water molecules. No electron density was observed for residues 1–4 or for the expression or purification tag. The Matthews' coefficient (Vm) is 2.06 Å3/Da, and the estimated solvent content is 40.0%. The Ramachandran plot, produced by MolProbity,8 shows that 97.0% and 3.0% of the residues are in favored and allowed regions, respectively. Crystal structure of AGT from Anabaena sp. (A) Stereo ribbon diagram of Anabena sp. AGT color-coded from N-terminus (blue) to C-terminus (red), showing the domain organization and location of the putative active site (PLP molecule shown in ball-and-stick representation). Helices H1–H15 and β-strands (β1–β12), as well as β-sheets A, B, and C are indicated. (B) Diagram showing the secondary structure elements in Anabaena sp. AGT superimposed on its primary sequence. The β-sheet designations are indicated by a red A, B, and C. Above each β-strand, β-bulges and γ-turns are indicated. β-hairpins are depicted as red loops. Disordered regions are depicted by a dashed line, with the corresponding sequence in brackets. The final refined structure of the AGT monomer consists of 12 β-strands (β1–β12), 12 α-helices (H1–H10, H12, H15), and 3 310-helices (H11, H13, H14) [Fig. 1(A and B)]. The total β-strand, α-helical, and 310-helical content is 13.3%, 45.9%, and 4.0%, respectively. The monomer consists of an extended N-terminal segment (residues 5–21), a large N-terminal domain (residues 22–282), and a smaller C-terminal domain (residues 283–381). The N-terminal domain has a 3-layer βαβ architecture and consists of 9 α-helices (H1–H9) and a 7-stranded (β2–β8), mixed β-sheet with 1765423 topology. The C-terminal domain is a 2-layer αβ structure with the α-helical face exposed at the protein surface, while the 3-stranded (β9, β11, β12), antiparallel β-sheet packs against the edge of the N-terminal domain [Fig. 1(A)]. An intimate dimer is found in the crystal, with an extensive interface formed by helices H3 and H4, loops 231–243, and 251–263 that connect helices H7, H8, and H9, and loop 207–217 between β-strands β6 and β7. Together these segments form a large flat surface, which packs against the equivalent region from the other monomer. In addition, the N-terminal extended segment (residues 5–21) of 1 monomer wraps around the other monomer, packing against helices H1 and H9 from the N-terminal domain [Fig. 2(A)]. The dimer interface buries a total of 3235 Å2 surface area per monomer. (A) Ribbon diagram of the Anabaena sp. AGT dimer. The N-terminal segment (red), N-terminal domain (green), and C-terminal domain (blue) are indicated for chain A. (B) Superposition of Anabaena sp. AGT (blue) and human AGT (PDB code: 1h0c; gray). The corresponding bound PLP and LLP (lysine-pyridoxal-5′-phosphate) ligands are shown in ball-and-stick representation, and regions of structural difference are labeled. (C) The PLP molecule bound to the active site of Anabaena sp. AGT and interacting residues are shown in ball-and-stick representation. 2Fo-Fc density for PLP contoured at 1σ is shown in blue. (D) Superposition of the active sites with the PLP and LLP molecules and interacting residues from Anabaena sp. AGT (blue) and their counterparts in human AGT (gray, residues labeled in brackets) in ball-and-stick representation. A structural similarity search, performed with the coordinates of 17130350 using the DALI server9 indicates structural similarity to the recently solved crystal structure of human AGT [Protein Data Bank (PDB) code: 1h0c],10 with a root-mean-square deviation (RMSD) of 1.7 Å over 319 aligned residues with 42% sequence identity [Fig. 2(B)]. A detailed structural comparison with human AGT reveals that the 2 structures are very similar. Small conformational differences are observed in the N-terminal segment (residues 5–16), in part of the loop (residues 252–256), between helices H8 and H9, and the two 310-helices, H11 (residues 308–316) and H13 (residues 346–355) [Fig. 2(B)]. Furthermore, the PLP molecule in human AGT is covalently linked to lysine 209, whereas the PLP in Anabaena sp. AGT is not [Fig. 2(C and D)]. The PLP in Anabaena sp. AGT is still located in the active site, but only hydrogen-bonded to lysine 209 NZ at a distance of 2.78 Å. We tested the possibility of a bound pyridoxamine phosphate (PMP) molecule by replacing PLP for PMP in the crystallographic refinement and found that it fits equally well in terms of difference density. However, the fact that the amino group (N4A) of PMP would be in H-bonding distance to lysine 209 NZ makes this scenario less likely. Therefore, we conclude that the ligand is a noncovalently linked PLP molecule that has not yet undergone Schiff-base formation. Similarly, a noncovalently-bound PLP molecule has also been observed in the structure of D-amino acid aminotransferase from Bacillus PS3.11 The structural similarity between Anabaena sp. and human AGT shows that, except for F108W and T83H [Fig. 2(D)], most of the active site residues including G82G are well conserved (also in mouse), which suggests that both AGTs share similar mechanisms. According to Fold and Function Assignment System (FFAS),12 the AGT family has hundreds of homologous sequences in pro- and eukaryotic proteomes. Models for AGT homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=17130350. The information reported here, in combination the human AGT (PDB: 1h0c)10 structure and further biochemical and biophysical studies, will yield valuable insights into the functional determinants of this protein and extend homology modeling of eukaryotic proteins. A putative AGT (GI: 17130350, Swiss-Prot: Q8YY48) was amplified by polymerase chain reaction (PCR) from genomic DNA from Anabaena sp. PCC 7120 (also referred to as Nostoc sp. PCC 7120, NC_003272) using Tag polymerase and primer pairs encoding the predicted 5′- and 3′-ends. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. Bacteria were lysed by sonication in lysis buffer [50 mM KPO4, pH 7.8, 300 mM NaCl, 10% glycerol, 5 mM imidazole, Roche ethylene diaminetetraacetic acid (EDTA)-free protease inhibitor tablets] with 0.5 mg/mL lysozyme. Immediately after sonication, the cell debris was pelleted by ultracentrifugation at 60,000 g for 20 min (4°C). The soluble fraction was applied to a gravity flow metal chelate column (Talon resin charged with cobalt; Clontech) equilibrated in lysis buffer. The column was then washed with 7 column volumes (CVs) of wash buffer (20 mM Tris, pH 7.8, 300 mM NaCl, 10% glycerol, 5 mM imidazole) and eluted with 3 CVs of elute buffer (25 mM Tris 7.8, 300 mM NaCl, 150 mM imidazole). The protein was then buffer exchanged into crystallization buffer (10 mM Tris, pH 7.8, 150 mM NaCl) and concentrated to 22 mg/mL by centrifugal ultrafiltration (Orbital). The protein was either frozen in liquid nitrogen for later use or used immediately for crystallization trials. The protein was crystallized using the nanodroplet vapor diffusion method13 with standard JCSG crystallization protocols.4 The crystallization solution contained 12% Polyethyleneglycol-monomethylether (PEG-MME) 5000, 0.2 M CaCl2, and 0.1 M N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) at pH 7.5. The cryosolution also contained 10% ethylene glycol. The crystals were indexed in the orthorhombic space group C2221 (Table I). Anomalous diffraction data were collected at the Advanced Photon Source (APS, Chicago, IL) on beamline 14BMC at a wavelength corresponding to the peak (λ1), of a selenium SAD experiment (Table I). The data sets were collected at 100 K using a MAR charge-coupled device (CCD) detector. Data were integrated and reduced using MOSFLM14 and then scaled with the program SCALA from the CCP4 suite.6 Data statistics are summarized in Table I. The data were processed using MOSFLM and the CCP4 suite,6 phased using SHELXD/E15 with 7 Se positions, and autotraced with ARP/wARP.16 Structure refinement was performed using REFMAC56 and O.17 Refinement statistics are summarized in Table I. The final model includes 1 protein monomer (residues 5–381), 1 PLP, and 423 water molecules in the asymmetric unit. No electron density was observed for residues 1–4, the side-chain atoms of R234, E297, K329, and Q378, or the expression and purification tags. Analysis of the stereochemical quality of the model was accomplished using the AutoDepInputTool (http://deposit.pdb.org/adit/), MolProbity,8 SFcheck 4.0,6 and WHATIF 5.0.18 The protein quaternary structure was analyzed using the PQS server (http://pqs.ebi.ac.uk/). Figure 1(B) was adapted from an analysis using PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/), and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of 17130350 have been deposited within the PDB and are accessible under the code 1vjo. 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. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences).