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• W2110442066 abstract "The Oxford Protein Production Facility (OPPF) was established to develop methods for high-throughput protein production and crystallization. As part of these developments, and in the context of the European SPINE project, a pilot study was undertaken on 48 proteins from Bacillus anthracis from protein families which were well conserved across a wide range of bacteria.1, 2 Bacillus anthracis, the causative agent of anthrax, is a large, gram-positive, spore-bearing bacterium. The genome of the Ames strain of the bacterium has been sequenced3 showing two plasmids, pXO1 and pXO2, that carry the major virulence factors, as well as normal chromosomal DNA of 5.23 megabases, predicted to code for about 5311 genes. The set of 48 proteins chosen for study1 are all encoded for by the chromosomal DNA. Annotation of the B. anthracis genome suggests it encodes for four or five 3-oxoacyl-[acyl carrier protein (ACP)] reductases. These form part of the β-ketoacyl-(ACP) reductase family, itself part of the short-chain dehydrogenase/reductase (SDR) superfamily, whose members catalyze a broad range of reactions using nucleotide cofactors. This study reports the structure of gene product BA3989, a 3-oxoacyl-(ACP) reductase of 246 residues, determined to a resolution of 2.4 Å using protein produced by the high-throughput pipeline of the OPPF. This enzyme performs the first reductive step in de novo fatty-acid biosynthesis4: the pyridine-nucleotide-dependent reduction of a 3-oxoacyl form of ACP (Fig. 1). Fatty-acid biosynthesis occurs by a series of universal biochemical transformations that are critical to almost all cells, but the pathway followed by bacterial systems differs substantially from that of higher organisms. Since this pathway is predicted to be essential5 it has been suggested as an attractive target for the development of novel antibiotics.6-8 Although to date no antibiotics target 3-oxoacyl-(ACP) reductases, a possible lead compound, epigallocatechin gallate from green tea, has been shown to be a potent inhibitor of the Escherichia coli oxoacyl reductase FabG.9 Schematic of the reaction catalyzed by 3-oxoacyl-(ACP) reductase. Cloning, expression and purification followed standard OPPF pipeline protocols.2, 10, 11 Briefly, the expression construct was generated by ligation-independent cloning using Gateway™ technology (Invitrogen). The BA3989 gene was amplified from genomic DNA with KOD HiFi™ polymerase (Novagen) using the forward primer 5′-ggggacaagtttgtacaaaaaagcaggcttcctggaagttctgttccagggcccgATGTTAAAAGGGAAAGTAGCATTAGTAACGGGC-3′ and the reverse primer 5′-ggggaccactttgtacaagaaagctgggtc tcaTTACATTACCATACCGCCATCAACATTTAACG-3′. The PCR product was purified using QIAquick 96 plates (Qiagen) and recombined with pDONR221 in the BP reaction. The insert from this vector was then transferred in the LR reaction to the expression vector pET15g which added a histidine purification tag and a 3C protease-cleavage site (shown by the arrow), MGSSHHHHHHSSGLVPRGSQSTSLYKKAGFLEVLFQ↓GP, to the N terminus of the full length protein.12 Recombinant LR clones were identified by PCR using a gene-specific forward primer, a T7 reverse primer and verified by sequencing. Protein was expressed in E. coli B834(DE3) cells grown at 37°C in GS96 media (QBiogene) to an A600 of 0.6, induced with isopropyl β-D-thiogalactopyranoside (IPTG) to 0.5 mM and then incubated for a further 20 h at 20°C. Cells were harvested by centrifugation at 6000g for 15 m and lysed using a Basic-Z Cell Disruptor (Constant Systems Ltd) at 30 Kpsi in 50 mM Tris pH 7.5, 500 mM NaCl, 20 mM Tris and 0.2% Tween-20. Soluble protein was purified by nickel-affinity chromatography followed by size-exclusion chromatography on an Äkta 3D™ (GE Healthcare). Protein-containing fractions were analysed by SDS-PAGE (Criterion-Biorad). The purification tag was removed by overnight incubation at 4°C with His-tagged 3C protease (prepared from pET-24/His-3C kindly provided by A. Geerlof, EMBL Heidelberg, Germany) with the protease and any uncleaved protein being removed by nickel-affinity chromatography. The protein was concentrated to 9.7 mg/mL using a 5 K MWCO Vivaspin 15 concentrator (Vivascience) in 20 mM Tris pH 7.5, 200 mM NaCl and 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Crystallization trials used the OPPF standard nanolitre crystallization protocol with standard OPPF screens.13-15 Crystals grew in Hampton Index condition 88: polyethylene glycol 3350 20% (w/v) and 0.2M tri-ammonium citrate pH 7.0 (Hampton Research). X-ray data were collected on station 14.1 at the Synchrotron Radiation Source (Daresbury, UK) from a crystal maintained at 100 K using an ADSC Quantum 4 detector. A total of 392 images were recorded using 0.5° oscillations, 20-s exposures and a crystal-to-detector distance of 135 mm. Prior to flash cooling, the crystal was protected by soaking briefly in perfluoropolyether XR-75 oil (Interchim). Data were indexed, integrated and reduced using DENZO and SCALEPACK16 (Table I). The structure was determined by molecular replacement using AMoRe17 and the tetramer of E. coli β-ketoacyl-(ACP) reductase (PDB ID 1Q7C; Ref.18) as the search model. Initial refinement and rebuilding used CNS19 and O,20 while the final cycles used REFMAC21 and Coot.22 Non-crystallographic restraints were kept tight throughout refinement, although restraints were removed from residues having different conformations in different chains (a total of 34 residues including the poorly ordered loop from 189–203), and TLS refinement was applied using each chain as a separate group. Structures were analyzed using MolProbity,23 DALI,24 and PISA.25 Structure superpositions were performed with SHP,26 the sequence alignment used ESPript27 and other figures were prepared using BobScript28 and Raster3D.29 Atomic coordinates (2uvd) and structure factors (r2uvdsf) have been deposited with the Protein Data Bank (PDB). The structure of a 3-oxoacyl-(ACP) reductase (EC 1.1.1.100) from B. anthracis has been determined to a resolution of 2.4 Å. The final model contains two tetramers displaying 222 symmetry (all chains are completely traced, although for some chains the electron density for residues 189–203 is poor) and 575 water molecules in the crystallographic asymmetric unit, but no bound cofactors or substrates. The model has a working R factor of 0.180 (free R factor of 0.225; Table I). Typically, there is a 0.2 Å root-mean-square (RMS) Cα-atom deviation between pairs of chains. The quality of the model (summarized in Table I) is good with an RMS deviation in bond lengths of 0.013 Å and 99.4% of residues in the allowed regions of the Ramachandran plot (as defined in MolProbity). Each chain comprises 10 α-helices (although helices α5 and α10 are kinked at residues Leu114 and Ser227, respectively) flanking a central seven-stranded parallelβ-sheet forming an NAD(P)-binding Rossmann-like domain typical of SDRs [Fig. 2(A,B)]. The DALI server reveals close structural similarity to many other SDRs, for example, a RMS deviation of 1.6 Å over 242 aligned Cα atoms for Magnaporthe grisea trihydroxynaphthalene reductase (PDB ID 1YBV; Ref.30; 37% sequence identity). The highest levels of sequence identity in the PDB are to E. coli 3-oxoacyl-(ACP) reductase (PDB ID 1I01; Ref.31; 56% identity) and C. thermocellum glucose-ribitol dehydrogenase (PDB ID 2HQ1; unpublished; 53% identity). However, these structures have no bound cofactors so the discussion below focuses on a comparison with the complex of Brassica napus β-keto-(ACP) reductase with NADP+ [PDB ID 1EDO; Ref.32; RMS deviation of 0.9 Å over 241 aligned Cα atoms using SHP; 52% sequence identity; Fig. 2(B,C)]. Structure of 3-oxoacyl-(ACP) reductase. (A) The overall structure of the monomer. The model is coloured from blue at the N-terminus to red at the C-terminus and secondary structural elements are labelled. (B) Sequence alignment of B. anthracis 3-oxoacyl-(ACP) reductase (top) with its homologue from Brassica napus. Conserved residues are highlighted in red and the catalytic Ser-Tyr-Lys triad is indicated by green triangles. Residue numbering and secondary structural elements relate to the B. anthracis enzyme. (C) A model for the active site of B. anthracis 3-oxoacyl-(ACP) reductase showing the proposed binding mode for NADP+. The position of the cofactor was inferred by superposition of the B. anthracis and B. napus (PDB ID 1EDO) structures. The secondary structure of the B. anthracis enzyme is shown in grey with residues predicted to interact with NADP+ shown as black sticks. NADP+ is shown as atom-coloured sticks with yellow carbon atoms and the catalytic triad is shown as atom-coloured sticks with green carbon atoms. (D) The proposed reaction mechanism showing the role of the catalytic triad in facilitating hydride transfer from NADPH. Analysis of the crystal packing (using PISA) indicates that, in common with other 3-oxoacyl-(ACP) reductases, the tetramer is the biologically active form. The tetramer measures ∼85 Å across and contains two types of dimerization interface. One interface (∼1600 Å2) comprises residues 98–107 (parts of helices α4 and α5) and residues 145–171 (parts of helices α6 and α7) from adjacent monomers. The interactions involving helices α4 and α5 are almost exclusively hydrophobic, although Lys115 makes salt bridges across to Glu103 and Asp107. In contrast, the interactions involving helices α6 and α7 contain several hydrogen bonds. The second interface (∼1350Å2) comprises the C-terminal regions (residues 166–246) of two monomers. In particular, residues 211–220 from one chain make a number of hydrogen bonds and salt bridges with residues 229–232 from the other. This structure of B. anthracis 3-oxoacyl-(ACP) reductase contains no bound cofactor/substrate, but the active site can be compared with the superposed structure of the NADP+/β-keto-(ACP) reductase complex from B. napus [Fig. 2(C)]. The hydrogen-bond network stabilizing the NADP+ would be expected to be similar to that of the B. napus complex. Based on this, the adenine ring would make direct hydrogen bonds between the nitrogen N1 and the amide hydrogen of Val63, and also between nitrogen N6 and the side chain of Asp62. The 3′ adenine ribose hydroxyl would form direct hydrogen bonds to the side chain of Ser13, the carbonyl oxygen of Gly11 and the amide hydrogen of Arg14 (whose side chain would be displaced on binding). The 2′ adenine ribose hydroxyl would hydrogen bond to the side chain of Ser13 and the phosphate group would interact with the side chain of Ser13 and the amide hydrogens of Gly37 and Asn38 (this loop is somewhat disordered in the B. anthracis structure and may only become properly ordered in the presence of the cofactor). The B. napus structure suggests that on binding NADP+, several waters, which occupy the putative pyrophosphate-binding pocket in our structure, would be displaced and the pyrophosphate moiety would form hydrogen bonds to main-chain atoms of Ile16, the side chain of Thr189 and, mediated by water, to the carbonyl oxygen of Arg14. The B. anthracis NADP+ binding pocket is ordered prior to binding, unlike the E. coli β-ketoacyl-(ACP)-reductase structure (PDB ID 1I01). The binding of the nicotinamide group would be stabilized by the protein main chain, whose conformation is largely similar to that observed in other NADP+/oxido-reductase complexes. A weak feature in our electron-density map appears to indicate the putative position of NO3*, which would make hydrogen bonds to the main chain of Asn89 and side chain of Lys158 (part of the catalytic triad). However, the region between residues 190 and 200 is poorly ordered, variable between the different chains in our model and differs significantly from the conformation found in NADP+−bound oxido-reductases. The side chain of Asp190 is positioned to interact with the pyrophosphate moiety, although for nicotinamide binding the residue would have to be displaced to prevent a steric clash. Based on the analysis of the B. napus structure and mechanistic studies33 the following mechanism is suggested [Fig. 2(D)]. In the first step, the phenolic group of residue Tyr154 forms a hydrogen bond with the carbonyl group of the 3-oxoacyl-(ACP). The hydroxyl group of Ser141 stabilizes this complex, helping to increase the polarization of the carbonyl group. The main role of Lys158 appears to be the stabilization of the nicotinamide ribose moiety of the NADPH through hydrogen bonds to the 2′-hydroxyl and 3′-hydoxyl groups. The NADPH is then involved in hydride transfer to the carbonyl carbon (becoming NADP+ in the process) and proton transfer from Tyr154 completes the reaction. Alternatively, Lys158 may be more actively involved in the reaction by forming part of a “proton relay” which helps replenish Tyr154 mediated by the 2′-hydroxyl of the nicotinamide ribose moiety of the NADP(H)." @default.
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• W2110442066 date "2007-09-25" @default.
• W2110442066 modified "2023-09-27" @default.
• W2110442066 title "Crystal structure of a 3-oxoacyl-(acylcarrier protein) reductase (BA3989) from Bacillus anthracis at 2.4-Å resolution" @default.
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• W2110442066 doi "https://doi.org/10.1002/prot.21624" @default.
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