Crystal structure of O‐acetylserine sulfhydrylase (TM0665) from Thermotoga maritima at 1.8 Å resolution (2004)

The TM0665 gene of Thermotoga maritima encodes a pyridoxal 5′-phosphate dependent O-acetylserine sulfhydrylase (EC 4.2.99.8) with a molecular weight of 31,131 Da and a calculated isoelectric point of 9.5. O-acetylserine sulfhydrylase (also known as cysteine synthase) catalyzes the last step in cysteine biosynthesis. The enzyme catalyzes the β-replacement of the acetyl group in O-acetyl-L-serine by sulfide to generate L-cysteine with production of acetate. Alternatively, the enzyme can use thiosulfate instead of sulfide to yield S-sulfo-L-cysteine. Here, we report the crystal structure of TM0665 determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics.1 The crystal structure of TM0665 [Fig. 1(A)] was determined to 1.8 Å resolution using the multiple-wavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes four protein molecules (residues 1–291), eight phosphate molecules, and 348 water molecules in the asymmetric unit. The Matthews' coefficient (Vm) for TM0665 is 2.55 Å3/Da and the estimated solvent content is 51.7%. The Ramachandran plot produced by PROCHECK 3.42 shows that 92% of the residues are in the most favored regions and 8% in additional allowed regions. No residues lie in the generously allowed or disallowed regions. A: Ribbon diagram of Thermotoga maritima TM0665 O-acetylserine sulfhydrylase color coded from N-terminus (blue) to C-terminus (red) showing the domain organization and location of the active site (arrow points towards bound phosphate). Helices (H1–H15) and β-strands (β1–β9) are indicated. The two phosphate molecules bound in the active site are shown in ball and stick. B: The TM0665 tetramer. C and D: Schematic representation of the interactions between TM0665 and the phosphate molecules bound to domain A (C) and domain B (D). Ligand bonds are in purple, protein amino acid bonds are in orange, and hydrogen-bonds are represented as dashed green lines with distances in A. Residues implicated in hydrophobic interactions are represented as barbed circle sections. The atoms are indicated as follows: carbon (black), nitrogen (blue), oxygen (red), phosphor (purple), and sulfur (yellow). The TM0665 monomer is composed of 15 helices (10 α-helices, and four 310-helices) and 9 β-strands [Fig. 1(A)]. The total α-helix, 310-helix, and β-strand content is 40.1%, 4.1%, and 15.4%, respectively. According to the Structural Classification of Proteins database (SCOP),3 TM0665 is a member of the “Tryptophan synthase beta subunit-like PLP-dependent enzyme” fold. Each monomer consists of two α/β domains (A and B) separated by a deep cleft and related by a pseudodyad. Domain A consists of 192 residues in two non-contiguous segments (1–35 and 135–291) and contains ten helices (H1 and H2, and H8 to H15) and six β-strands (β1, β2, and β6 to β9), arranged as a six-stranded β-sheet with a -1 -2X -1X -1X 3X topology. Strands β1 and β2 form a β-hairpin the end of which (26–34) contains a peculiar β-bulge at Asn28, that is also conserved in the TM1255 homologue from Salmonella typhimurium (PDB: 1D6S).8 Domain B consists of the 99 amino acids that encompass residues 36 to 134. Domain B comprises five helices (H3 to H7) and three β-strands (β3 to β5) organized as a parallel β-sheet [Figs. 1(A) and 2(A)]. A: Diagram showing the secondary structure elements in TM0665 superimposed on its primary sequence. The location of the β-hairpin formed by β-strands 1 and 2 is also depicted. B: Location of the phosphate 1 and phosphate 2 in the active site of TM0665 O-acetylserine sulfhydrylase. C: Location of pyridoxal 5′-phosphate (yellow) and methionine (magenta) in the catalytic site of O-acetylserine sulfhydrylase from Salmonella typhimurium (PDB: 1d6s). A phosphate ligand is bound to each domain. The interactions between the phosphate ligands and surrounding residues in the active site of the enzyme are depicted in Figure 1(C) and 1(D). Phosphate-2 is located in the pyridoxal 5′-phophate binding site in domain A, and mimics the phosphate group of pyridoxal 5′-phosphate. Phosphate-1 localizes to the substrate binding site in domain B and mimics the carboxyl group of the amino acid substrate (Figure 2B and 2C). Five TM0665 paralogs are present in the Thermotoga proteome: TM0138, tryptophan synthase; TM0225, putative 1-aminocyclopropane-1-carboxylate deaminase; TM0356, threonine dehydratase; TM0539, tryptophan synthase; and TM0546, threonine synthase. All of these proteins are involved in amino acid biosynthesis, with the exception of TM0225. TM0665 shares the same fold with tryptophan and threonine synthases. Homology structural models for a number of TM0665 orthologs and paralogs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM0665 The crystallographic packing in the TM0665 structure suggests that a tetramer is the biologically-relevant oligomeric form with a buried surface area of 2,802 Å2 per monomer. The prediction about the tetrameric nature of the O-acetylserine sulfhydrylase from Thermotoga maritima [Fig. 1(C)] is consistent with literature sources which indicate that the enzyme forms a homo-dimer or homo-tetramer. Tetrameric O-acetylserine sulfhydrylases have been previously observed in Saccharomyces cerevisiae,4 Salmonella typhimurium,5 and Arabidopsis thaliana6 among others. A structural similarity search, performed with the DALI server7 with the coordinates of TM0665, indicates that the closest structural homologue is O-acetylserine sulfhydrylase from Salmonella typhimurium (PDB: 1D6S).8 The RMSD between TM0665 and O-acetylserine sulfhydrylase is 1.2 Å over 302 residues with 52% sequence identity. The structure reported here represents the first structure of a hyperthermophilic O-acetylserine sulfhydrylase, whose structure has been determined by X-ray crystallography using the MAD method. We expect that the information reported here will yield valuable insights into the determinants for catalysis, substrate specificity, and thermal stability of this protein family. O-acetylserine sulfhydrylase (TIGR: TM0665; Swissprot: Q9WZD3) was PCR amplified using Pfu (Stratagene) from Thermotoga maritima strain MSB8 genomic DNA with primer pairs encoding the predicted 5′- and 3′-ends of TM0665. The PCR product was cloned into plasmid pMH2T7, which encodes an expression and purification tag consisting of the residues 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 after a freeze-thaw procedure in Lysis Buffer (50mM Tris pH 7.9, 50mM NaCl, 1mM MgCl2, 0.25mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), 1mg/ml lysozyme) and cell debris pelleted by centrifugation at 3600× g for 60 min. The soluble fraction was applied to a nickel chelate resin (Pharmacia) previously equilibrated with Equilibration Buffer (50 mM KH2PO4 pH 7.8, 0.25 mM TCEP, 10% v/v glycerol, 0.3 M NaCl) containing 20 mM imidazole. The resin was washed with Equilibration Buffer containing 40 mM imidazole, and protein eluted Elution Buffer (20 mM Tris pH 7.9, 10% glycerol, 0.25mM TCEP, 300 mM imidazole). Buffer exchange was performed to remove imidazole from the protein eluate, and the protein in Buffer Q (20 mM Tris pH 7.9, 50 mM NaCl, 5% v/v glycerol, 0.25 mM TCEP) was then applied to a Resource Q column (Pharmacia). Protein was eluted using a linear gradient of up to 500 mM NaCl. Appropriate fractions were buffer exchanged into Crystallization Buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 TCEP). The protein was concentrated to 13 mg/mL for crystallization assays by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method9 with standard JCSG crystallization protocols.1 The crystallization buffer contained 1.8 M NaK2PO4 at pH 6.9. The crystals were indexed in the monoclinic space group P21 (Table I). Native and multi-wavelength anomalous diffraction data were collected at SSRL (Stanford, USA) on beamline 9-2 at multiple wavelengths using the BLU-ICE10 data collection environment (Table I). All data sets were collected at 100 K using a Quantum 4 CCD detector. Data were integrated and reduced using Mosflm11 and then scaled with the program SCALA from the CCP4 suite.12 Data statistics are summarized in Table I. The structure was determined using the software packages SnB,13 the CCP4 suite12 and SOLVE.14 Structure refinement was performed using CNS and Refmac5.12, 15 The asymmetric unit contains four molecules. Refinement statistics are summarized in Table I. The final model for protein chain A contains the native sequence plus residue His0 from the purification tag. For protein chain B, the final model also lacks the purification tag (except His0) and residues Pro106 to Lys112. For protein chain C, the model lacks the purification tag (except His0), residues Leu102 to Gly126, and the C-terminal Leu291. Finally, for protein chain D, the final model lacks the purification tag (except His0 and His-1) and residues Leu104 to Lys117. Analysis of the stereochemical quality of the models was accomplished using the JCSG Validation Central suite, which integrates seven validation tools: Procheck 3.5.4, SFcheck 4.0, Prove 2.5.1, ERRAT, WASP, DDQ 2.0, and Whatcheck. The Validation Central suite is accessible at http://www.jcsg.org. Atomic coordinates of the final model and experimental structure factors of TM0665 have been deposited with the PDB and are accessible under the code 1o58. This work was supported by NIH Protein Structure Initiative grant P50-GM 62411 from the National Institute of General Medical Sciences (www.nigms.nih.gov). 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).