Báo cáo khoa học: X-ray crystallographic and enzymatic analyses of shikimate dehydrogenase from Staphylococcus epidermidis pot

X-ray crystallographic and enzymatic analyses of shikimate dehydrogenase from Staphylococcus epidermidis Implications for substrate binding and conformational change Cong Han1,*, Tiancen Hu2,*, Dalei Wu2, Su Qu1, Jiahai Zhou3, Jianping Ding4, Xu Shen2, Di Qu1 and Hualiang Jiang2 Institutes of Biomedical Sciences and Key Laboratory of Medical Molecular Virology, Institute of Medical Microbiology, Shanghai Medical College, Fudan University, China Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China State Key Laboratory of Molecular Biology and Research Center for Structural Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China Keywords crystal structure; shikimate dehydrogenase; shikimate pathway; site-directed mutagenesis; Staphylococcus epidermidis Correspondence D Qu, Institutes of Biomedical Sciences and Key Laboratory of Medical Molecular Virology, Institute of Medical Microbiology, Shanghai Medical College, Fudan University, Shanghai 200032, China Fax: +86 21 54237603 Tel: +86 21 54237524 E-mail: dqu@shmu.edu.cn X Shen, Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China Fax ⁄ Tel: +86 21 50806918 E-mail: xshen@mail.shcnc.ac.cn *These authors contributed equally to this work Database Coordinate and structure factor files for SeSDH and SeSDH in complex with shikimate have been deposited in the Protein Data Bank under the accession numbers 3DON and 3DOO Shikimate dehydrogenase (SDH) catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway In this study, we determined the kinetic properties and crystal structures of Staphylococcus epidermidis SDH (SeSDH) both in its ligand-free form and in complex with shikimate SeSDH has a kcat of 22.8 s)1 and a Km of 73 lm towards shikimate, and a Km of 100 lm towards NADP The overall folding of SeSDH comprises the N-terminal a ⁄ b domain for substrate binding and the C-terminal Rossmann fold for NADP binding The active site is within a large groove between the two domains Residue Tyr211, normally regarded as important for substrate binding, does not interact with shikimate in the binary SeSDH–shikimate complex structure However, the Y211F mutation leads to a significant decrease in kcat and a minor increase in the Km for shikimate The results indicate that the main function of Tyr211 may be to stabilize the catalytic intermediate during catalysis The NADP-binding domain of SeSDH is less conserved The usually long helix specifically recognizing the adenine ribose phosphate is substituted with a short 310 helix in the NADP-binding domain Moreover, the interdomain angle of SeSDH is the widest among all known SDH structures, indicating an inactive ‘open’ state of the SeSDH structure Thus, a ‘closing’ process might occur upon NADP binding to bring the cofactor close to the substrate for catalysis (Received 28 August 2008, revised November 2008, accepted 12 December 2008) doi:10.1111/j.1742-4658.2008.06856.x Abbreviations AaSDH, Aquifex aeolicus shikimate dehydrogenase; Af, Archaeoglobus fulgidus; AtSDH, Arabidopsis thaliana shikimate dehydrogenase; EcSDH, Escherichia coli shikimate dehydrogenase; Gk, Geobacillus kaustophilus; HiSDH, Haemophilus influenzae shikimate dehydrogenase; HpSDH, Helicobacter pylori shikimate dehydrogenase; IPTG, isopropyl thio-b-D-galactoside; MAD, multiple-wavelength anomalous diffraction; MjSDH, Methanococcus jannaschii shikimate dehydrogenase; MtSDH, Mycobacterium tuberculosis shikimate dehydrogenase; SDH, shikimate dehydrogenase; SDHL, shikimate dehydrogenase-like enzyme; SeMet-SeSDH, selenomethionine-substituted SeSDH; SeSDH, Staphylococcus epidermidis shikimate dehydrogenase; TtSDH, Thermus thermophilus shikimate dehydrogenase FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS 1125 Shikimate dehydrogenase from S epidermidis C Han et al Staphylococcus epidermidis is the most common causal microorganism responsible for infections of implanted medical devices such as central venous catheters, cardiac pacemakers, artificial lenses and prosthetic joints [1] The pathogenesis of S epidermidis-mediated infections is mainly attributed to the adherence and subsequent formation of a multilayered biofilm of S epidermidis on biomaterials Bacterial cells within the biofilm are dramatically less susceptible to antibiotic treatment and attacks by the immune system than planktonic cells Moreover, a biofilm may continuously release bacteria into the bloodstream on a chronic basis, resulting in bacteremia Therefore, the formation of a biofilm of pathogenic bacteria often results in the removal of implanted medical devices, thus leading to substantial morbidity and mortality [2] Moreover, the appearance of multiresistant and vancomycinresistant S epidermidis strains may impair the efficacy of antibiotic treatment regimens The pressing need to control S epidermidis-mediated infection is creating an urgent challenge to discover novel antibacterial agents that are active against new bacterial targets In bacteria, seven enzymes involved in the shikimate pathway catalyze the sequential conversion from erythrose 4-phosphate and phosphoenolpyruvate via shikimate to chorismate, which serves as a precursor for the synthesis of essential metabolites such as aromatic amino acids, folic acid and ubiquinone [3] The shikimate pathway is crucial in algae, higher plants, bacteria, apicomplexan parasites and fungi, but is absent in mammals, making the enzymes involved in this pathway potential targets for the development of nontoxic antimicrobial agents, herbicides and anti-parasite drugs [4] Shikimate dehydrogenase (SDH, EC 1.1.1.25) catalyzes the fourth reaction of the shikimate pathway, an NADPH-dependent reduction of 3-dehydroshikimate to shikimate The inhibitors targeting Helicobacter pylori SDH can block growth of the bacteria in vitro, demonstrating that SDH is a promising target for antimicrobial agents [5] Shikimate dehydrogenase belongs to the superfamily of NAD(P)H-dependent oxidoreductases In plant, SDH is coupled with 3-dehydroquinate dehydratase to form a bifunctional enzyme [6] In fungi and yeast, SDH serves as a component of the penta-functional AROM enzyme complex that catalyzes steps 2–6 within the shikimate pathway [7] There are three SDH orthologues – AroE, YdiB and SDH-like enzyme (SDHL) – in bacteria AroE has been identified as a single monofunctional enzyme that is strictly specific for the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in most bacteria YdiB, found in Escherichia coli, Salmonella typhi1126 murium, Streptococcus pneumoniae and Haemophilus influenzae, is characterized as a quinate ⁄ shikimate dehydrogenase that not only retains the function of AroE but also reversibly reduces dehydroquinate to quinate using either NADH or NADPH as a cofactor It plays a more important role in the quinate pathway than in the shikimate pathway The SDHL, in a small group of species such as Pseudomonas, only catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate but with a dramatically lower catalytic rate than that of AroE [8] However, the complete genome sequence of S epidermidis has revealed the presence of only AroE in the shikimate biosynthetic route A total of 20 crystal structures of SDH have been determined so far covering all the three orthologues of SDH mentioned above, including AroE from E coli (PDB code: 1NYT [9]), H influenzae (1P74 and 1P77 [10]), Methanococcus jannaschii (1NVT [11]), Aquifex aeolicus (2HK7, 2HK8 and 2HK9 [12]), Thermus thermophilus (1WXD, 2CY0, 2D5C and 2EV9 [13]), Arabidopsis thaliana (2GPT [14], 2O7Q and 2O7S [15]) and Geobacillus kaustophilus (2EGG); YdiB from E coli (1O9B [9], 1NPD [16] and 1VI2) and Corynebacterium glutamicum (2NLO [17]); and SDHL from H influenzae (1NPY [8]) These structures comprise the following diverse conformations (a) apo-enzyme (1NPY, 1P74, 1WXD, 2EGG, 2HK7, 2HK8 and 2NLO), (b) binary complex bound with either cofactor (1NPD, 1NVT, 1NYT, 1O9B, 1P77, 1VI2 and 2CY0) or substrate (2D5C, 2GPT and 2O7Q) and (c) inactive (2HK9_A and 2EV9) and active (2HK9_D and 2O7S) ternary complexes Analysis of these different conformations reveals the binding information of substrate and cofactor, the structural basis underlying the cofactor specificity of AroE and YdiB [9], and the putative catalytic mechanism of SDH [12] Notably, the relative positions of the two domains responsible for substrate and cofactor binding, respectively, are different among these structures, representing two distinct states of SDH, namely the open form and the closed form Only the closed form is believed to be competent for catalysis [9,12,13] In addition to the unliganded enzyme structures, the crystallographic analysis of SDH in complex with cofactor, and of even ternary enzyme–cofactor–substrate complexes, shedsnew light on the catalytic mechanism and provides clues for the rational design of anti-infective compounds Although the 3D structures of SDH have offered much detailed structural information, few reported SDH structure originates from pathogenic bacteria, particularly gram-positive bacteria FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS C Han et al In this article, we report the crystal structures of S epidermidis SDH (SeSDH), in both ligand-free form and in complex with shikimate, and the enzymatic characterization of SeSDH Our structure represents the first SDH structure from gram-positive bacteria The overall folding of SeSDH is similar to that of other SDH structures, constituted by the N-terminal a ⁄ b domain for substrate binding and the C-terminal Rossmann fold for NADP binding The active site is present within a large groove between these two domains The N-terminal domain and the shikimatebinding residues of SeSDH are highly conserved among SDH enzymes, except that the tyrosine residue (Tyr211), normally regarded as important for substrate binding, does not interact with shikimate in the crystal structure of SeSDH in complex with shikimate On the basis of the enzymatic data of the Y211F mutant, we suggest that Tyr211 plays a crucial role to stabilize the catalytic intermediate during catalysis The NADPbinding domain of SeSDH is less conserved A long helix specifically recognizing the adenine ribose phosphate is substituted with a short 310 helix in this domain Moreover, the interdomain angle of SeSDH is the widest among all known SDH structures Extensive comparison with other SDH structures indicates an inactive ‘open’ state of our structure and implies that a ‘closing’ process might occur upon NADP binding to bring the cofactor close to the substrate for catalysis Our study is expected to enhance the understanding of SDH features and provide useful information for the rational drug design of novel antimicrobial agents targeting SeSDH Shikimate dehydrogenase from S epidermidis (Fig 1) Considering that the molecular mass of SeSDH is equal to 31 011 Da, we conclude that SeSDH might exist as a monomer in solution state Next, we investigated the catalytic properties of SeSDH, as well as its Y211F mutant, and the effects of pH on catalysis The kinetic parameters Km and Vmax were calculated from the slope and intercept values of the linear fit in a Lineweaver–Burke plot For example, the Lineweaver–Burke plot for the NADPdependent oxidation of shikimate to 3-dehydroshikimate is shown in Fig In comparison with the kinetic parameters of SDH enzymes from other bacteria shown in Table 1, the Km and kcat values of SeSDH were comparable with those of A aeolicus SDH (AaSDH) [12] and H pylori SDH (HpSDH) [5] As illustrated in Table 1, the Y211F mutation resulted in a 345-fold decrease in the kcat value, a three-fold increase in the Km value and a 1073-fold decrease in the kcat ⁄ Km value for shikimate, which indicates that Tyr211 plays a major role in the catalytic process and a minor role in the initial substrate binding Similarly to HpSDH [5] and Archaeoglobus fulgidus SDH (Af- Results Biochemical characterization of SeSDH and its Y211F mutant After one-step purification of nickel-affinity chromatography, the recombinant SeSDH, coupled to a C-terminus six-histidine tag, was purified to apparent homogeneity The LC-ESI-MS spectral data gave a molecular mass of 31 011 Da for the recombinant SeSDH, which is in good agreement with the theoretical molecular mass of 31 013 Da calculated from the amino acid sequence Similarly, the substitution of Y211F was corroborated by MS The predicted and observed molecular mass values were 30 996 and 30 995 Da, respectively In the gel-filtration experiments, the size-exclusion chromatography of SeSDH showed only one peak The elution volume of SeSDH was larger than that of ovalbumin (43.0 kDa) and smaller than that of chymotrypsinogen A (25.0 kDa) Fig Gel-filtration study of SeSDH (A) Size-exclusion chromatography of low-molecular-mass standards The elution points of molecular mass standards [BSA (67.0 kDa), ovalbumin (43.0 kDa), chymotrypsinogen A (25.0 kDa) and ribonuclease A (13.7 kDa)] are shown for reference (B) Size-exclusion chromatography of SeSDH FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS 1127 Shikimate dehydrogenase from S epidermidis C Han et al Fig pH profile of SeSDH enzyme activity Fig The Lineweaver–Burke plot for the NADP-dependent oxidation of shikimate to 3-dehydroshikimate SDH) [18], SeSDH can also oxidize shikimate using NAD as a cofactor, yielding a kcat of 87 ± 20 s)1, a Km of 10.6 ± 2.6 mm and a kcat ⁄ Km of 8.2 · 103 m)1Ỉs)1 towards NAD SeSDH showed a Km for NAD that was almost 100 times higher than that for NADP at the saturation of shikimate, suggesting that NADP is the preferred cofactor of SeSDH We also tested whether SeSDH could utilize quinate as a substrate Even in the presence of a high concentration of quinate (4 mm), SeSDH displayed no activity using either NADP or NAD as a cofactor The pH can dramatically affect enzyme activity in a number of ways As shown in Fig 3, SeSDH is active within a wide pH range of 7–12, with the highest activity occurring at around pH 11 It was reported that the pH optimum of HpSDH is 8–10 [5], and the pH optimum of AfSDH is 7–7.5 [18] By contrast, SeSDH exhibits very high activity at an extremely basic pH range of 10–12, similar to Mycobacterium tuberculosis SDH (MtSDH) [19] Thus, it can be speculated that the active site of SDH might involve several acidic ⁄ basic amino acid residues that play crucial roles in the substrate-binding and catalytic processes Overall 3D structure of SeSDH The overall structure of apo-SeSDH is basically identical to that of the shikimate–SeSDH complex, with ˚ an rmsd value of 0.51 A from aligning 246 pairs of Ca atoms Both structures contain one molecule per asymmetric unit, indicating that SeSDH might function as a monomer, which is also in accordance with gel-filtration analysis However, M jannaschii SDH (MjSDH) and T thermophilus SDH (TtSDH) are considered to be dimers under physiological conditions [11,13] The statistics of the apo-SeSDH and shikimatebound SeSDH structures are summarized in Table The apo-SeSDH structure contains 264 amino acids (residues 1–271, the loop containing residues 185–191 is disordered) and 223 water molecules The binary shikimate–SeSDH structure contains 258 amino acids Table Comparison of kinetic parameters of SDHs from various bacteria SDH species pH kcat (s)1) (shikimate) Km (lM) (shikimate) Km (lM) (NADP) kcat ⁄ Km (M)1Ỉs)1) (shikimate) SeSDH Y211F mutant AaSDHa HpSDHb AfSDHc MtSDHd EcSDHe 8.0 8.0 9.0 8.0 7.3 9.0 9.0 22.8 0.066 55.5 7.7 361 399 237 73 227 42.4 148 170 30 65 100 279 42.4 182 190 63 56 3.12 · 105 291 1.31 · 106 5.2 · 104 2.12 · 106 1.33 · 107 3.65 · 106 ± ± ± ± 1.5 0.017 1.5 0.9 ± ± ± ± ± 15 1.6 28 30 ± ± ± ± ± 10 55 0.9 27 10 kcat ⁄ Km (M)1Ỉs)1) (NADP) 2.28 236 1.31 3.9 1.9 6.33 4.23 · 105 · · · · · 106 104 106 106 106 Kinetic parameters for A aeolicus SDH are from [12] b Kinetic parameters for H pylori SDH are from [5] c Kinetic parameters for A fulgidus SDH are from [18] d Kinetic parameters for M tuberculosis SDH are from [19] e Kinetic parameters for E coli SDH are from [9] a 1128 FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS C Han et al Shikimate dehydrogenase from S epidermidis (residues 1–267, the loop containing residues 185–193 is disordered), one shikimate molecule and 151 water molecules As illustrated in Fig 4A, similarly to other SDH structures, SeSDH comprises two domains The N-terminal substrate-binding domain contains amino acid residues 1–100 and 233–267 It is formed by a central six-strand mixed b sheet (b2, b1, b3, b5, b6 and b4; b5 is antiparallel to the others) flanked by three a-helices (a1, a9 and a8) on the inner side and by two a-helices (a2 and a3) and two 310 helices (g1 and g2) on the outer side Helices a8 and a9 are in the most C-terminal region of the polypeptide, which folds back into the N-terminal domain The C-terminal NADP-binding domain comprises two parallel Rossmann folds The mostly parallel six-stranded b-sheet (b9, b8, b7, b10, b11 and b12) at the core of the domain is flanked by three helices (a4, a5 and g4) on the inner side and by two helices (g5 and a6) on the outer side The two domains are connected by two a-helices (a4 and a8) in the middle of the molecule, creating a deep groove where the catalysis occurs Substrate-binding domain Superposition reveals that the overall folding of the SeSDH substrate-binding domain is highly similar to that of other SDH structures, with rmsd values rang˚ ing from 0.68 to 1.6 A The only difference is that the a2 helix in SeSDH is packed more towards the central b-sheet, and the orientation of the C-terminal helix a9 is divergent among these enzymes (Fig 4B) The substrate shikimate is unambiguously positioned in the well-defined annealed omit map of the complex Table Data collection, phasing, and refinement statistics Apo-SeSDH Data collection Space group Cell dimensions ˚ a, b, c (A) a, b, c (°) ˚ Wavelength (A) ˚ )a Resolution (A Rsymb I ⁄ rI Completeness (%) Redundancy Refinement ˚ Resolution (A) No reflections Rwork ⁄ Rfreec No of atoms Protein Water Substrate B-factors Protein Water Substrate ˚ rmsd (A) Bond angles (°) Ramachandran plot (%) Most favored Allowed Generously allowed Disallowed Shikimate-SeSDH Apo-SeMet-SeSDH P212121 P21 P21 P21 P21 52.88, 54.15, 102.72 90, 90, 90 45.19, 52.53, 56.78 90, 96.14, 90 45.15, 52.36, 56.71 90, 95.77, 90 Peak 45.18, 52.38, 56.74 90, 95.77, 90 Edge 45.20, 52.40, 56.77 90, 95.72, 90 RemoteH 1.5418 50–2.1 (2.18–2.10) 0.100 (0.334) 5.9 (2.0) 97.9 (97.9) 5.57 (5.82) 1.5418 30–2.2 (2.28–2.20) 0.122 (0.380) 5.2 (1.9) 99.2 (99.2) 3.52 (3.45) 0.97901 20–2.5 (2.56–2.50) 0.036 (0.055) 22.4 (16.4) 95.8 (74.7) 2.89 (2.54) 0.97953 20–2.5 (2.56–2.50) 0.037 (0.059) 21.9 (15.7) 95.7 (74.7) 2.90 (2.55) 0.96429 20–2.5 (2.56–2.50) 0.040 (0.064) 20.8 (14.8) 95.7 (75.6) 2.90 (2.55) 15–2.1 16 488 0.183 ⁄ 0.260 15–2.2 12 793 0.188 ⁄ 0.264 2091 223 – 2048 151 12 22.3 31.0 – 0.02 1.72 28.1 31.5 25.5 0.03 2.08 93.6 6.4 0 89.0 11.0 0 P P P P Values in parentheses are for the highest-resolution shell b Rsym = h i jIhi À hIh ij= h i Ihi , where Ihi and hIh i are the ith and mean P P c measurement of the intensity of reflection h, respectively Rwork ⁄ Rfree = h jFo:h À Fc:h j= h Fo:h , where Fo.h and Fc.h are the observed and calculated structure factor amplitudes, respectively a FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS 1129 Shikimate dehydrogenase from S epidermidis C Han et al A B C D Fig The overall structure and the substrate-binding domain of SeSDH in complex with shikimate (A) The overall structure of SeSDH is shown as a cartoon The N-terminal substrate-binding domain is colored in orange and green, and the C-terminal NADP-binding domain is colored in blue The bound shikimate molecule and its binding residues are shown as stick and lines, respectively (B) Superposition of the N-terminal domains from all SDH structures The structure of SeSDH is colored in blue (C) Schematic diagram of the substrate-binding site of SeSDH Dotted lines represent hydrogen bonds The asterisk beside the C3-hydroxyl group of shikimate indicates the proton to be delivered to the bulk solvent during catalysis The proton-conducting route is represented by the arrows The inset is the annealed omit map around shikimate, contoured at the 1.0r level (D) Superposition of the substrate-binding residues from all SDH structure The structure of SeSDH is colored in cyan, and the distance between the side chain of Tyr211 and the carboxyl of shikimate is colored in red structure contoured at 1.0r (Fig 4C, inset) The bound molecule adopts a half-chair conformation, and the group bonded to C3 is orthogonal to the ring system As illustrated by the scheme (Fig 4C) and the structure-based sequence alignment (Fig 5), the shikimate is hydrogen bonded to several highly conserved residues In detail, the carboxylate group of shikimate is recognized by the hydroxyl groups of Ser13 and Ser15, as well as by the backbone amide of Leu14 via a water molecule The C5-hydroxyl group of shikimate forms hydrogen bonds with the side-chain amides of Asn58 and Asn85 The C4-hydroxyl group of shikimate interacts with the carboxylate group of Asp100 and with the side-chain amides of Asn85 and Lys64 The C3-hydroxyl group forms extensive hydrogen bonds with the side chains of Lys64, Asp100, Thr60 and Gln239 The absolutely conserved residues Lys64 and Asp100 are also believed to be catalytically active and responsible for the deprotonation of the C3-hydroxyl group during the catalysis [12] In brief, Fig Structure-based sequence alignment of various SDHs The secondary structures are from SeSDH a-helices are represented as squiggles, 310 helices are marked with g, b-strands are rendered as arrows and b-turns are shown as TT The blue and green numbers beneath the alignment indicate substrate-binding residues and NADP-binding residues, respectively The parenthesized 211 indicates that the conserved residue Tyr211 does not interact with the substrate in the SeSDH structure The figure was prepared using the program ESPript The sequence alignment was created using the following sequences from the Protein Data Bank: SeSDH(3DON), GkSDH(2EGG), MjSDH(1NVT), EcSDH(1NYT), TtSDH(1WXD), AaSDH (2HK8), HiSDH(1P74), AtSDH(2GPT), EcYdiB(1NPD), CgYdiB(2NLO) and HiSDHL(1NPY) 1130 FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS C Han et al FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS Shikimate dehydrogenase from S epidermidis 1131 Shikimate dehydrogenase from S epidermidis C Han et al Lys64 serves as a general base to abstract a proton from the C3-hydroxyl group and transfer it to the adjacent Asp100, which subsequently delivers the proton to the bulk solvent via structurally conserved water molecules to refresh the enzyme The proton-conducting route is represented by the arrows in Fig 4C The conserved residue Tyr211 participates in substrate binding in the other structures of SDH in complex with shikimate, and is believed to be the ionizable group with a pKa of 9.7 that must be protonated for catalysis [12] The distances between the conserved residue, Tyr211, and shikimate in the active sites of various SDH structures are shown in Table Remarkably, the phenol hydroxyl of Tyr211 is not within hydrogen bond distance from the carboxyl ˚ oxygen of shikimate (5.7 A) in the binary SeSDH– shikimate complex structure (Fig 4D) However, the Y211F mutation results in a remarkable reduction in enzyme activity, which indicates that the phenol hydroxyl of Tyr211 still interacts with shikimate to stabilize the catalytic intermediate, playing an essential role in the catalytic process NADP-binding domain The C-terminal NADP-binding domain of SeSDH is less conserved than the substrate-binding domain The rmsd values obtained from superposing various SDH ˚ NADP-binding domains ranged from 0.91 to 5.84 A Figure 6A shows the structural superposition of the NADP-binding domains from SeSDH and AaSDH ˚ Table The distance (A) between the conserved tyrosine residue and shikimate in the active sites of various SDH structures Tyr-OH – the nearest carboxyl Tyrosine Tyr-Ca – oxygen of numbering shikimate-C1 shikimate Binary complex (shikimate) SeSDH AtSDH (2GPT_A) AtSDH (2O7Q_A) TtSDH (2D5C_A) TtSDH (2D5C_B) Ternary complex AaSDH (2HK9_D, active) AaSDH (2HK9_A, inactive) AtSDH (2O7S_A, active) TtSDH (2EV9_A, inactive) a 211 550 550 207 207 12.7 9.1 9.1 10.6 8.7 5.7 2.5 2.5 2.8 6.4a 216 216 550 207 8.1 9.3 8.9 11.2 2.5 2.8 2.7 3.7 The tyrosine has flipped its side chain to interact with the 3-hydroxyl of shikimate via a water molecule Its Ca atom still remains close to the carboxyl of shikimate 1132 The latter represents the common fold of NADP-binding domain in SDH structures There are three obvious differences observed from the superposition First, the long helix interacting with the adenine ribose 2-phosphate of NADP in AaSDH is substituted with a short 310 helix (g4) in SeSDH Remarkably, the short helix has a high temperature factor in both ligand-free ˚ ˚ (46.3 A2 versus the overall 22.3 A2) and shikimate˚ ˚ versus the overall 28.1 A2) struccomplexed (36.7 A tures, indicating the flexibility of the region The ‘basic patch’ for binding the adenine ribose phosphate (NRTXXR ⁄ K motif, residue 148–153), which endows SDH with the specificity for NADP over NAD [9], is located at the helix g4 and at the nearby b8–g4 loop Second, a helix packing at the outer side of the central b-sheet in AaSDH is replaced with two short helices (a6 and g5) in SeSDH Third, the part in SeSDH corresponding to the b10–b11 loop of AaSDH is disordered This region in AaSDH is found immediately after the residue that helps to sandwich the NADP adenine, and thus the flexibility of this part in SeSDH is probably caused by the absence of NADP However, despite these differences, the key NADP-binding motifs are still structurally conserved in all reported SDH structures (Fig 6A, inset), including the basic patch, the adenine sandwich, the nicotinamide-binding residues and the glycine-rich loop (GAGGA motif, residue 124–128) recognizing the pyrophosphate and the adenine ribose 3¢-hydroxyl of NADP Based on this observation, we modeled the NADP molecule from the superposed AaSDH structure into the C-terminal domain of SeSDH to check whether the conformation of this domain is appropriate for binding NADP As shown in Fig 6B, the glycine-rich loop and the nicotinamide-binding residues, as well as Asn148 and Thr150 within the basic patch, are in the correct positions to form hydrogen bonds with their binding partners of NADP However, the basic patch residue Arg149 deviates away from the adenine ribose phosphate, whereas Arg153 collides with it Furthermore, the two residues Arg149 and Pro184 flanking the adenine are too far away from each other to form a sandwich structure As the nicotinamide nucleotide and the pyrophosphate of NADP are properly anchored in the C-terminal domain of SeSDH, the adenosine moiety is unlikely to adopt a different orientation, implying that the basic patch and the adenosine sandwich structures of SeSDH might undergo conformational change upon NADP binding Actually, the loop following residue Pro184 is completely disordered in both ligand-free and shikimate-complexed SeSDH structures, further indicating the flexibility of the region Similarly, NADP-induced conformational changes are indicated FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS C Han et al Shikimate dehydrogenase from S epidermidis A B Fig The NADP-binding domain of SeSDH (A) The superposition of the NADP-binding domains of SeSDH (cyan) and AaSDH ternary complex (orange) The NADP molecule from AaSDH is shown as a stick The inset represents the superposition of the NADP-binding domains from all SDH structures SeSDH is colored in cyan The conserved NADP-binding motifs are colored in blue (B) The NADP molecule from the superposed AaSDH is modeled into the NADP-binding domain of SeSDH The residues colored in cyan are not in the correct positions to interact with NADP The inset is the superposition of apo-(yellow) and NADP-bound (cyan) HiSDH structures The putative conformational change occurring upon NADP binding is indicated by the arrow by the structures of apo-H influenzae shikimate dehydrogenase (HiSDH) and its complex with NADPH (Fig 6B, inset) [10] Open and closed conformational change Two overall structural states of the SDH structure – the open form and the closed form – have been reported [9,12,13] Table summarizes the key features of various SDH structures The ‘openness’ of the molecule could be evaluated by the interdomain angle, which is defined as the angle among the centroids of the two domains and the Ca atom of the conserved hinge residue aspartate (Asp100 in SeSDH) As shown in Table 4, the interdomain angle of SeSDH is the widest among all reported SDH structures, indicating that the structure represents the most ‘open’ form of SDH There are two distinct structural features related to such ‘openness’ of SeSDH First, the Ca distance between the central glycine of the conserved NADP-binding motif GAGGA (Gly126 in SeSDH) and the catalytic lysine residue (Lys64 in ˚ SeSDH) is $ 14 A, much larger than the correspond˚ ing distances in the active ternary complexes ($ A) ˚ and the reported ‘closed’ structures (< 11.2 A) It is FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS 1133 Shikimate dehydrogenase from S epidermidis C Han et al Table Key features of various SDH structures Interdomain angle (°) SeSDH SeSDH AaSDH (2HK9_D)c AaSDH (2HK9_A) AaSDH (2HK8_F) AtSDH (2O7S) TtSDH (2EV9_A) TtSDH (2CY0_B) EcSDH (1NYT_A) EcSDH (1NYT_C) HiSDH (1P77_A) HiSDH (1P74_A) GkSDH (2EGG_A) MjSDH (1NVT_A) Native Shikimate Ternary (active) closedd Ternary (inactive)e undefined Native, open Ternary (active) undefined Ternary (inactive) closed Native, open NADP, closed NADP, open NADP, undefined Native, undefined Native, undefined Native, undefined Gly126 fi Lys64a Tyr211 fi Ser13 Tyr211 fi Ser15 BCD-NDb ⁄ Boverall (%) 157.8 164.7 127.3 139.3 149.4 120.9 134.5 142.2 146.8 139.9 153.8 144.2 147.9 132.9 14.2 14.4 8.5 9.8 12.1 8.2 11.2 12.5 8.4 12.2 12.2 14.6 13.7 13.5 13.3 13.9 9.3 9.5 11.3 9.1 11.3 11.7 10.7 13.0 11.6 13.6 13.3 12.6 14.3 14.9 10.9 11.3 12.5 11.4 13.1 13.6 12.7 14.5 13.1 15.0 14.7 13.8 30.1 32.3 21.9 15.5 7.2 42.6 )29.3 14.0 16.2 0.6 0.0 )0.8 24.3 41.5 ˚ The distance (A) between the Ca atoms of the two residues in SeSDH or their counterparts in the other structures b The difference between the B factor of the N-terminal domain and that of the C-terminal domain c The PDB code and chain name d The reported closed ⁄ open state e The structure contains a disordered NADP nicotinamide and thus is considered inactive a also larger than those in most of the ‘open’ and native forms of SDH The feature indicates that the NADPbinding motif is far away from the shikimate-binding site in the SeSDH structure, representing an unfavorable state for the hydride transfer between NADP and shikimate Thus, we conclude that a closing process between the two domains of SeSDH might occur upon NADP approaching shikimate during catalysis Second, the Ca distances between the conserved tyrosine residue (Tyr211 in SeSDH) and the two serine residues interacting with the carboxyl of shikimate (Ser13 and Ser15 in SeSDH) are larger than those in other SDH structures, indicating that Tyr211 of SeSDH does not interact with substrate in the structure To investigate in greater detail the open–close mechanism of SeSDH, we superposed the substrate-binding domain of the SeSDH structure with that of the active AaSDH ternary complex structure As shown in Fig 7, the NADP-binding domain of SeSDH is located further away from the substrate-binding domain than that of AaSDH The deviation starts from the conserved hinge residue Asp100 at the beginning of helix a4 in SeSDH, which coincides with the centroid of the molecule (Fig 7A) Superposition of the closed and open forms of AaSDH by their substrate-binding domains reveals that the departure of their NADP-binding domains also begins from the corresponding conserved residue Asp106 (Fig 7A, inset) Detailed inspection of the two superposed NADP-binding domains shows that the key NADPbinding motifs in SeSDH deviate from those of 1134 AaSDH in the same direction (Fig 7B–D), which implies that these motifs of SeSDH might be able to ‘pivot’ to their competent NADP-binding positions upon the closure of the molecule via the hinge residue B-factor analysis of all the 14 SDH structures listed in Table also indicated that the temperature factor of the NADP-binding domain is usually higher than that of the substrate-binding domain, especially at the helices containing the NADP-binding motifs, suggesting that the NADP-binding domain of SDH might be more prone to conformational changes than the substrate-binding domain Discussion To date, many studies of SDH have revealed various intriguing parts of the medically important target enzyme [8–16], including 20 crystal structures of various SDHs in their ligand-free form and in complex with either substrate or cofactor or both, enzymatic kinetics for catalysis, a detailed catalytic mechanism of SDH underlying the hydride transfer between NADP and substrate coupled with the deprotonation of the 3-hydroxyl group of shikimate and the transfer route of the abstracted proton to the bulk solvent [12], and the relationship between the catalytic activity and the open–closed conformations of SDH The catalytic properties and substrate specificity of SeSDH demonstrate that SeSDH belongs to the AroE enzyme family and can utilize NAD as a cofactor in vitro The pH profile of SeSDH indicates that the basic condition may be suitable for the ionization of the catalytic FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS C Han et al Shikimate dehydrogenase from S epidermidis b A B C D Fig The structure superposition of SeSDH complexed with shikimate (cyan) and the AaSDH ternary complex (orange) by their N-terminal domains (A) View from the back of the superposed a4 helices The hinge residue Asp100 is shown as a stick The centroids are represented as spheres SKM represents shikimate The inset shows the superposition of the open (magenta) and closed (orange) forms of AaSDH (B–D) The comparison of the key NADP-binding motifs from SeSDH and AaSDH residues, for the binding affinity of substrate and for the stability of the enzyme The crystal structures of the ligand-free and shikimate-complexed SeSDH offer incremental knowledge for understanding the catalytic mechanism of SDH, especially the role of the conserved residue Tyr211 formerly regarded important for substrate binding, the conformational change that might occur upon NADP binding and the requirement FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS 1135 Shikimate dehydrogenase from S epidermidis C Han et al of a ‘closing’ process during catalysis as well as its putative underlying mechanism The overall folding of SeSDH is similar to that of other SDH structures, comprising an N-terminal substrate-binding domain and a C-terminal NADPbinding domain On the basis of gel-filtration and crystallographic analyses, we conclude that SeSDH exists as a monomer in both crystal and solution states The substrate-binding domain of SeSDH is structurally conserved in all three classes of SDH The substrate forms hydrogen bonds with several polar residues in the domain Structure-based sequence alignment (Fig 5) shows that most hydroxyl-binding residues are conserved among all three SDH classes, including Thr60, Asn85, Gln239 and the two catalytic active residues Lys64 and Asp100 The carboxyl-binding residues Ser13, Ser15 and Tyr211 are conserved in both AroE and YdiB, but not in SDHL The most noteworthy residue is Tyr211 As shown in Table 3, in all structures of SDH complexed with shikimate (except for SeSDH), the tyrosine residue is hydrogen bonded to the carboxyl oxygen of shikimate By contrast, in the current structure of SeSDH complexed with shikimate, the tyrosine residue is too far away from the substrate to form a hydrogen bond The Y211F mutation leads to a significant decrease in the kcat value by a factor of 345 but a minor increase in the Km value for shikimate by a factor of The above results taken together indicate that the main function of Tyr211 may be to stabilize the catalytic intermediate during catalysis Thus, a ‘closing’ process of SeSDH is expected to occur upon NADP binding, which might lead to the binding of the Tyr211 residue to shikimate In fact, we may find that the Ca distances between the tyrosine residue and the other two shikimate-carboxyl˚ binding serine residues ($ and $ 11 A, respectively), in the two confirmed active conformations of SDH [AaSDH and A thaliana shikimate dehydrogenase (AtSDH)], are substantially smaller than those in other SDH structures (Table 4), which further supports our speculation The NADP-binding domain of SeSDH is less structurally conserved than the substrate-binding domain, especially in view of the fact that a common long helix bearing a basic patch for binding the adenine ribose phosphate is substituted with a small 310 helix g4 in SeSDH Moreover, compared with the NADP-bound C-terminal domain of AaSDH, we found that the domain of SeSDH will be appropriate for binding NADP if some small conformational changes occur These changes included the side-chain swing of the two arginine residues constituting the basic patch and the 1136 closing-up of the adenine sandwich Actually, such NADP-induced conformational changes are indicated by the HiSDH structures [10], and are also suggested by homology modeling and CD of MtSDH [20] The high temperature factor values of the SeSDH NADP-binding domain also imply their predisposition to conformational changes By contrast, the structure of SeSDH complexed with shikimate is almost the same with its apo form, indicating that no conformational change could be induced by substrate alone It has been widely accepted that SDH has two overall conformations, namely the open form and the closed form, and the closed form is necessary for catalysis [9,12,13] Michel et al suggested that E coli shikimate dehydrogenase (EcSDH) switches from the open to the closed conformation upon substrate binding [9] Bagautdinov et al reported that both forms of TtSDH exist in solution, and the cofactor only binds to the closed form while the substrate binds to both forms Thus, they suggested a random Bi–Bi reaction mechanism of TtSDH [13] The structure of SeSDH represents the most open form of all known SDH structures, as indicated by its largest interdomain angle (Table 4) Accordingly, the ˚ distance ($ 14 A) between the NADP binding ‘GAGGA’ motif and the catalytically active residue Lys64 in SeSDH is the largest among all SDH structures, almost twice as large as those in the active ternary ˚ complexes ($ A) Such a distance is directly proportional to the interdomain angle of the enzyme Thus, we could expect the occurrence of a ‘closing’ process of SeSDH, probably via the hinge residue, Asp100, to bring NADP close to the substrate for catalysis The binding of shikimate alone does not cause any significant structural change of SeSDH, and the C-terminal domain is more prone to rearrangement than the N-terminal domain in SeSDH (as reflected by the temperature factor) Thus, we believe that the ‘closing’ process might be induced by NADP binding and stabilized by shikimate binding In summary, we determined the crystal structures and enzymatic properties of SeSDH The combination of the crystallographic and enzymatic analysis sheds more light on the role of key amino acid residues and the catalytic mechanism Notably, the Y211F mutation proves that Tyr211 plays a vital role for the oxidation of shikimate, although it has no interaction with shikimate in the crystal structure of the SeSDH–shikimate complex We speculate that the open to closed conformational change of SeSDH might occur upon NADP binding, which needs to be identified by analysis of the ternary complex structure of SeSDH–shikimate–NADP(H) FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS C Han et al Experimental procedures Materials S epidermidis strain RP62A was obtained from the American Type Culture Collection All chemicals and reagents were of reagent grade or ultrapure quality, and are commercially available Preparation of wild-type and mutant SeSDH Based on the genome sequence of S epidermidis strain RP62A (GenBank accession number: NC_002976), two PCR primers (forward: 5¢-GCGCATCCATATGAAATTT GCAGTAATTGG-3¢ and reverse: 5¢-CCGCTCGAGTAA TTCTCCTTTCAATTTTTG-3¢) were designed to amplify the aroE gene on the chromosome of S epidermidis strain RP62A The PCR products were digested using restriction endonucleases NdeI and XhoI (Fermentas, Burlington, Ontario, Canada) and then cloned into a prokaryotic expression vector pET22b (Novagen, Madison, WI, USA) to produce the recombinant plasmid pET22b–SeSDH, containing a C-terminal six-histidine tag, for purification purposes The recombinant clone pET22b–SeSDH was verified by DNA sequencing The recombinant clone pET22b–SeSDH was transformed into E coli strain BL21(DE3) (Stratagene, La Jolla, CA, USA) and cultured at 37 °C in Luria–Bertani (LB) medium supplemented with 100 lgỈmL)1 of ampicillin When the A600 reached 0.6, the culture was induced by 0.4 mm isopropyl thio-b-d-galactoside (IPTG) and incubated at 25 °C for an additional h The cells were harvested by centrifugation and suspended in buffer A (20 mm Tris ⁄ HCl, pH 8.0, 500 mm NaCl, 10 mm imidazole) After sonication on ice, the mixture was centrifuged to yield a clear supernatant, which was loaded onto a column containing Ni-nitrilotriacetic acid resin (Qiagen, Hilden, Germany) pre-equilibrated in buffer A The column was washed with buffer B (20 mm Tris ⁄ HCl, pH 8.0, 500 mm NaCl, 20 mm imidazole) for several times and eluted with buffer C (20 mm Tris ⁄ HCl, pH 8.0, 500 mm NaCl, 200 mm imidazole), then the eluted fractions were pooled and dialyzed against buffer D (20 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl) for enzymatic assay and crystallization trials The samples were pooled and concentrated by ultrafiltration using an Amicon centrifugal filter device All purification, dialysis and concentration procedures were performed at °C Protein concentration was determined by the Bradford assay using BSA as standard The oligomeric state was studied by gel-filtration chromatography using an AKTA purifier system (GE Healthcare, Uppsala, Sweden) The sample of protein was chromatographed on a HiLoad 16 ⁄ 60 Superdex 75 prepacked grade column with buffer D at a flow rate of mLỈmin)1 The Shikimate dehydrogenase from S epidermidis Superdex 75 column was precalibrated with the following low-molecular-mass standards: BSA (67.0 kDa), ovalbumin (43.0 kDa), chymotrypsinogen A (25.0 kDa) and ribonuclease A (13.7 kDa) (GE Healthcare, Uppsala, Sweden) Site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit (Stratagene) using the plasmid pET22b–SeSDH as template The primers for the Y211F mutant were: (sense) 5¢-GTAAGTGATATTGT TTTTAATCCATATAAAACACC-3¢ and (antisense) 5¢-G GTGTTTTATATGGATTAAAAACAATATCACTTAC-3¢ The mutant plasmid was confirmed by DNA sequencing before transformation into E coli strain BL21(DE3) The expression and purification of the Y211F mutant were the same as that of the wild-type enzyme Preparation of selenomethionyl SeSDH Selenomethionine-substituted SeSDH (SeMet-SeSDH) was generated in the methionine auxotrophic E coli strain B834(DE3) (Novagen) The strain transformed with pET22b–SeSDH was grown at 37 °C in M9 minimal medium, which contained 40 mgỈL)1 of all of the amino acids except methionine A selenomethionine (Acros, Geel, Belgium) stock solution of 10 mgỈmL)1 in water was added to the medium to a concentration of 40 mgỈL)1 The SeMet-SeSDH protein was induced with 0.4 mm IPTG for about 12 h, and purified using the same method as for the native protein Finally, the SeMet-SeSDH fractions were pooled and dialyzed against buffer E (20 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl, mm dithiothreitol and 0.2 mm EDTA) for crystallization trials ES-MS analysis of the SeMet-SeSDH indicated substitution of all six methionine residues Enzymatic activity assay The enzymatic activities of SeSDH and its Y211F mutant were assayed at 25 °C by monitoring the reduction of NADP (or NAD) at 340 nm (e340 = 6180 m)1Ỉcm)1) in the presence of shikimate All assays were conducted in a 96-well microplate spectrophotometer (Beckman Coulter DTX880, Fullerton, CA, USA) The assay mixture contained shikimate and NADP (or NAD) at the desired concentrations in 100 mm Tris–HCl (pH 8.0) The Km and Vmax values for substrate and cofactor were determined by varying the concentrations of one ligand while keeping the other at saturation In the experiment where shikimate was varied (0.0625, 0.125, 0.25, 0.5, and mm), the concentration of NADP was maintained at mm, whereas the concentration of shikimate was fixed at mm when NADP was varied (0.0625, 0.125, 0.25, 0.5, and mm) The assay reaction was initiated by the addition of diluted enzyme solution To measure the kinetic parameters for NAD, the concentration of shikimate was fixed at mm when NAD FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS 1137 Shikimate dehydrogenase from S epidermidis C Han et al was varied (0.5, 1, 2, 4, and 16 mm) The kinetic parameters Km and Vmax were calculated from the slope and intercept values of the linear fit in a Lineweaver–Burke plot To test the enzymatic activity of SeSDH in the presence of quinate, the assay solution consisted of 100 mm Tris ⁄ HCl (pH 8.0), mm quinate and mm NADP (or NAD) Each measure was taken in triplicate The effects of pH on the enzymatic activity of SeSDH were determined using the above assay method All the assay solutions contained mm shikimate and mm NADP The enzymatic kinetics of SeSDH was measured in buffers of different pH values (50 mm Bistris ⁄ HCl for pH 5.0–7.0, Tris ⁄ HCl for pH 8.0–9.0 and Caps ⁄ NaOH for pH 10–12) All assays were conducted three times Crystallization and data collection All crystallization trials were performed at 277 K using the hanging-drop vapor-diffusion method Initial crystals of SeSDH were obtained using commercially available crystallization screen kits (Hampton Research Crystal Screens and 2, Aliso Viejo, CA, USA) After several rounds of optimization, crystals of both native SeSDH and SeMet-SeSDH were grown in drops containing equal volumes of the protein solution (20 mgỈmL)1) and the crystallization solution (0.1 m tri-sodium citrate dihydrate at pH 5.8, 24% PEG4000, 0.2 m ammonium acetate) to the maximal size after 30 days The crystals were harvested and cryoprotected with the reservoir solution containing up to 10% (v ⁄ v) glycerol To generate the SeSDH–shikimate complex, protein samples were incubated with mm shikimate at 277 K for 12 h prior to crystallization Crystals of SeSDH complexed with shikimate were obtained using a different crystallization buffer (0.1 m sodium cacodylate at pH 6.5, 23% PEG 8000, 0.2 m ammonium sulfate) The crystals of SeSDH in complex with shikimate were cryoprotected by including 30% (v ⁄ v) glycerol in the mother liquor Each crystal was picked up with a nylon loop and flash-cooled in liquid nitrogen before data collection The crystal diffraction data for the crystals of both apo-SeSDH and SeSDH complexed with shikimate were collected at 100 K on a Rigaku rotating-anode X-ray generator operated at ˚ 50 kV and 100 mA (k = 1.5418 A) with a Rigaku R-AXIS IV+ imaging-plate detector The data were processed + using the crystalclear program [21] For the crystal of SeMet-SeSDH, a multiple-wavelength anomalous diffraction data set was collected on beamline BL-6A of the Photon Factory (Tsukuba, Japan) with an ADSC Quantum 4R ˚ CCD detector using three wavelengths (peak = 0.97901 A; ˚ ; and high-energy remote = 0.96429 A) ˚ edge = 0.97953 A The three wavelengths were determined by a fluorescence scan for selenium The multiple-wavelength anomalous diffraction data were processed using the HKL-2000 suite of programs [22] The statistics of data collection are summarized in Table 1138 Structure solution and refinement For phase determination, SOLVE was used to locate all of the six selenium sites and to calculate the phases [23] RESOLVE was used for density modification and initial model building [24] The complete model was built using COOT [25] Refinement of the structure was carried out using CNS [26] and Refmac from the CCP4 suite [27] procheck was used to check the stereochemical quality of the final structural model [28] All figures and superpositions were made using pymol [29] Centroids of the structures were calculated using PDBSET (http://www ccp4.ac.uk/html/pdbset.html) The crystal structures of apo-SeSDH and of SeSDH in complex with shikimate were solved by molecular replacement using the SeMet-SeSDH structure as a search model The refinement statistics are summarized in Table The atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession numbers 3DON and 3DOO Acknowledgements We thank the staff members at the Photon Factory of Japan for technical support in diffraction data collection, Dr Yanhui Xu for assistance with gel-filtration experiments and Dr Xinhua Ji for critical reading of the manuscript This work was supported by the State Key Program of Basic Research of China (grant 2002CB512803), the International Science and Technology Cooperation Program of China (grant 2006DFA32760), the National Natural Science Foundation of China (grant 30800036), and China Postdoctoral Science Foundation Funded Project (grants 200801172 and 20060400574) References Vuong C & Otto M (2002) Staphylococcus epidermidis infections Microbes Infect 4, 481–489 Costerton JW, Stewart PS & Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections Science 284, 1318–1322 Parish T & Stoker NG 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WL (2002) The PyMOL Molecular Graphics System DeLano Scientific, Palo Alto, CA FEBS Journal 276 (2009) 1125–1139 ª 2009 The Authors Journal compilation ª 2009 FEBS 1139 ... FEBS Shikimate dehydrogenase from S epidermidis 1131 Shikimate dehydrogenase from S epidermidis C Han et al Lys64 serves as a general base to abstract a proton from the C3-hydroxyl group and transfer... oxidation of shikimate to 3-dehydroshikimate SDH) [18], SeSDH can also oxidize shikimate using NAD as a cofactor, yielding a kcat of 87 ± 20 s)1, a Km of 10.6 ± 2.6 mm and a kcat ⁄ Km of 8.2 ·... structures of S epidermidis SDH (SeSDH), in both ligand-free form and in complex with shikimate, and the enzymatic characterization of SeSDH Our structure represents the first SDH structure from gram-positive