Báo cáo khoa học: Crystal structures of open and closed forms of D-serine deaminase from Salmonella typhimurium – implications on substrate specificity and catalysis pptx

Crystal structures of open and closed forms of D-serine deaminase from Salmonella typhimurium – implications on substrate specificity and catalysis Sakshibeedu Rajegowda Bharath 1 , Shveta Bisht 1 , Handanhal Subbarao Savithri 2 and Mattur Ramabhadrashastry Narasimha Murthy 1 1 Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India 2 Department of Biochemistry, Indian Institute of Science, Bangalore, India Keywords D-serine deaminase; open and closed conformations; pyridoxal 5¢ phosphate dependent Foldtype II enzyme; X-ray diffraction; a, b elimination Correspondence M. R. N. Murthy, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India Fax: +91 80 2360 0535 Tel: +91 80 2293 2458 E-mail: mrn@mbu.iisc.ernet.in (Received 12 March 2011, revised 29 May 2011, accepted 7 June 2011) doi:10.1111/j.1742-4658.2011.08210.x Metabolism of D-amino acids is of considerable interest due to their key importance in cell structure and function. Salmonella typhimurium D-serine deaminase (StDSD) is a pyridoxal 5¢ phosphate (PLP) dependent enzyme that catalyses degr adation of D-Ser to pyruvate and ammonia. The fi rst crystal structure of D-serine deaminase described here reveals a typical Foldtype II or tryptophan synthase b subunit fold of PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild-type StDSD (WtDSD) and selenomethionine labelled StDSD (SeMetDSD), significant electron density was not observed for the cofactor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two struc- tures. The WtDSD was in an open conformation while SeMetDSD, crystal- lized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other Foldtype II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modelling suggests that Thr166 may be involved in abstraction of proton from the Ca atom of the substrate. Apart from the physiological reaction, StDSD catalyses a, b elimination of D-Thr, D-Allothr and L-Ser to the corresponding a-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differ- ences in the rate of reaction with these substrates. Introduction Pyridoxal 5¢ phosphate (PLP) dependent enzymes constitute a diverse family of proteins involved in the metabolism of amino acids, amino sugars and amino group containing lipids. A majority of them are key enzymes in the metabolism of amino acids. The reac- tions catalysed include the transfer of amino group, decarboxylation, inter-conversion of l- and d-amino acids and removal or replacement of chemical groups at a, b or c positions [1]. Functionally, PLP-dependent enzymes have been classified into three groups ( a, b and c families) based on the carbon atom at which the net reaction takes place [2]. Structurally, they have been classified into five groups [3–6]: Foldtype I enzymes that belong to the aspartate aminotransferase Abbreviations DNPH, 2,4-dinitrophenyl hydrazine; EcDSD, D-serine deaminase from Escherichia coli; LSD, L-serine dehydratase; PLP, pyridoxal 5¢-phosphate; SeMetDSD, selenomethionine D-serine deaminase; SpSR, Schizosaccharomyces pombe serine racemase; StDSD, Salmonella typhimurium D-serine deaminase; TRPSb, tryptophan synthase b; WtDSD, wild-type D-serine deaminase. FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS 2879 family, Foldtype II that resemble tryptophan synthase b, Foldtype III that are related to the alanine racemase family, Foldtype IV enzymes related to the d-amino acid aminotransferase family and Foldtype V or glyco- gen phosphorylase family. Most of the amino acid dehydratases belong to the tryptophan synthase b (TRPSb) family or Foldtype II PLP-dependent enzymes [5]. These enzymes catalyse irreversible degradation of amino acids to the respec- tive a-keto acids. l-serine and l-threonine dehydratases have been purified and characterized both structurally and biochemically from several organisms [7–9]. Comparison of the amino acid sequence of d-serine deaminase from Salmonella typhimurium (StDSD) with those of other PLP-dependent enzymes suggests that it belongs to the Foldtype II or TRPSb family, although the sequence identities are low (15–23%). However, there are d-serine deaminases (unrelated in sequence to StDSD) which are annotated in sequence databases as Foldtype III or as members of the alanine racemase family. d-Serine deaminase from Escherichia coli (EcDSD; EC 4.3.1.18) exhibits b-elimination activity with d-Ser, d-Thr, d-Allothr and l-Ser with a pH optimum of 8.0 [10]. In most bacteria, DSD probably acts as a detoxi- fying enzyme, carrying out degradation of d-Ser. Most organisms fail to survive and propagate on d-Ser containing nutrient media. This is attributed to the formation of d-Ser activated aminoacyl tRNA leading to toxicity and retardation of cell growth. d-Ser is a co-agonist of NMDA channel receptors and therefore EcDSD has been routinely included in the purification of NMDA receptors from organotypic hippocampal slices [11]. The Saccharomyces cerevisiae DSD has been used in diagnostic laboratories for quantitative deter- mination of d-Ser in human brain and urine [12]. The E. coli dsdA gene has been found to be an excellent marker for construction of strains for which the use of antibiotic resistance genes as selective markers is not allowed [13,14]. EcDSD (48 kDa) [15] and Klebsiella DSD (46 kDa) [16] are known to be functional as monomers in contrast to the majority of Foldtype II PLP-dependent enzymes, which are dimers. However, DSD is a dimer of 118 kDa in S. cerevisiae [17] and a heterodimer of 40 and 40.4 kDa subunits in chicken [18]. EcDSD has been found to be activated by NH þ 4 and K + and to a lesser extent by Na + ions [10,19]. It was proposed that K + is not involved directly in catalysis but is required for stabilizing the active site geometry [20]. Although crystals of EcDSD suitable for X-ray diffraction studies have been obtained [21], its structure has not been reported in the literature. Comparative studies on monomeric StDSD and the more common dimeric forms of Foldtype II PLP-dependent enzymes will allow examination of the plausible role of oligomeric state in these enzymes [22]. In this paper, we report the first crystal structure of a Foldtype II d-serine deaminase and describe features of the active site essential for catalysis. The crystal structures reported are wild-type StDSD (WtDSD) and selenomethionine incorp orated StDS D (SeM etDSD) crystallized in the presence of isoserine. Although nei- ther of the structures had density corresponding to the cofactor, WtDSD was in an open conformation while SeMetDSD was in the closed conformation, suggesting that StDSD exhibits a domain movement similar to those of rat liver l-serine deaminase (rat liver LSD) [9] and serine racemase from Schizosaccharomyces pombe (SpSR) [23]. Examination of the monomeric structure of StDSD suggests that a dimeric structure similar to those of other Foldtype II PLP-dependent enzymes would lead to unacceptable van der Waals contacts involving segments of StDSD that are insertions with respect to other Foldtype II enzymes. Electron density for a putative Na + ion was located close to the active site of SeMetDSD but not of WtDSD. The active site geometry allows identification of residues that may play a key role in catalysis. Results and Discussion Biochemical studies on StDSD Recombinant StDSD was expressed in E. coli as a hexa-histidine tagged protein and purified by nickel nitrilotriacetic acid affinity and size exclusion chroma- tography. The purified protein was yellow in colour with an absorbance maximum at 415 nm, indicating the presence of PLP as an internal aldimine. As in several other PLP-dependent enzymes, a small peak at 340 nm was also observed. The A 280 ⁄ A 415 ratio was close to 10. The peak at 415 nm was independent of pH in the range 6.0–9.0. However, a small increase in the peak at 330–340 nm was observed close to pH 6.0. Similar observations have been reported for the EcDSD by Dupourque et al. [10]. The peaks at 415 and 330 nm have been attributed to the ketoenamine and enolimine forms, respectively, of the internal aldi- mine [24]. StDSD was most active with d-Ser. The activities of WtDSD (2.09 lmolÆmg )1 Æmin )1 ) and SeMetDSD (2.12 lmolÆmg )1 Æmin )1 ) with d-Ser as the substrate were comparable. It has been reported that the pres- ence of Na + or K + ions enhances the activity of EcDSD [20,24]. The enzymatic properties of StDSD Crystal structure of D-serine deaminase S. R. Bharath et al. 2880 FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS were therefore examined in the presence of Na + or K + ions and the results are tabulated (Table 1). K m and V max for d-Ser were about twice as high in the presence of Na + than with K + . In the presence of Na + , K m for d-Ser was higher than that of d-Thr. However, V max was an order of magnitude higher for d-Ser. In contrast, in the presence of K + ions, K m was lower and V max was higher for d-Ser than for d -Thr. The enzyme was much less active with d-Allothr and l-Ser. With both d-Thr and d-Allothr, the enzyme was more active in the presence of K + than Na + . Structure and model quality Crystal structures of SeMetDSD (1.9 A ˚ ) and WtDSD (2.4 A ˚ ) were solved using four-wavelength anomalous dispersion (4W-MAD) and molecular replacement methods respectively. The data collection and structure refinement statistics are given Tables 2 and 3 respec- tively. In WtDSD, except for two short stretches (68–71 and 234–239) electron density is of good quality throughout the polypeptide main chain. In SeMetDSD, electron density is absent for only two C-terminal residues (439–440). A total of 17 and seven residues have been truncated according to the extent of observed electron density in WtDSD and SeMetDSD, respectively. In both structures, the residues forming the C-terminal hexa-histidine tag were not included in the model due to absence of a well-defined electron density. In SeMetDSD, 94.4% and 4.7% of residues were in favoured and additionally allowed regions, respectively, of the Ramachandran plot [25,26]. One residue (Ile111) was in the disallowed region. The WtDSD structure had 93.1% and 6.3% of the residues in the favoured and additionally allowed regions and two residues (Ile111 and His319) in the disallowed region. Statistics of the Ramachandran plot obtained from procheck [27] are given in Table 3. Interestingly, a well-defined density for PLP was not observed in the two structures. The polypeptide fold of StDSD is illustrated in Fig. 1. The secondary structural elements have been assigned using dssp [28]. As in other PLP-dependent enzymes of Foldtype II, the StDSD monomer consists of a small domain (residues 43–75, 109–238) and a large domain (residues 1–42, 76–108 and 239–440). The small domain folds as an open twisted a ⁄ b structure consist- ing of a four-stranded (S4–S7) parallel b-sheet sand- wiched between one helix (H11) on the solvent facing side and two helices (H9 and H10) on the other side. Four more helices (H4, H5, H7 and H8) occur in this domain. The core of the large domain contains a seven- stranded mixed b-sheet surrounded by eight helices on the solvent facing side (H6, H17, H18, H19 and H20 on one side and H1, H2 and H3 on the other side) and six helices that occur between the two domains (H12, H13, H14, H15, H16 and H21). In the central b-sheet of the large domain, all except two short strands at the peri- phery (S1 and S2) are parallel. The strands of the cen- tral b-sheet are strongly twisted. The N-terminal helices H1 and H2 protrude away from the large domain. StDSD structure was used as the template for identi- fying other proteins with similar folds in the Protein Data Bank (PDB) using the program dali [29] with the view of identifying shared and unique structural fea- tures of StDSD. There were 245 hits with Z-scores higher than 20.0, of which 36 were unique structures. The top hits corresponded to threonine deaminase, serine racemase and l-serine dehydratase. Although the overall fold of StDSD is similar to those of the PLP- dependent Foldtype II enzymes, the helices H1, H2, H3 and H19 and the antiparallel b strand S1 in the large domain and the helices H5 and H8 in the small domain are significantly different and could be considered as additions to the fold of the TRPSb family. These struc- tural segments are shown in orange in Fig. 1. Gel filtration studies indicate that StDSD is a mono- mer in solution. This is consistent with the earlier results obtained with EcDSD [10,15]. However, it is in contrast to most of the enzymes belonging to the TRPSb family, which are dimers in solution (except for threonine synthase from E. coli (PDB code 1VB3) and yeast (PDB code 1KL7), which are also mono- mers). Modelling of StDSD resembling the dimeric structures of other Foldtype II PLP-dependent enzymes such as O-acetylserine sulfhydrylase (OASS) or cystathionine b-synthase suggests that H5, H6 and H21 prevent dimer formation by causing steric clashes. Comparison between WtDSD and SeMetDSD The rmsd upon superposition of corresponding Ca atoms of WtDSD and SeMetDSD polypeptides is Table 1. Kinetic parameters of StDSD with various substrates, phosphate buffer pH 7.5. V max is expressed as micromoles of pyru- vate formed per milligram of protein per minute of reaction. Substrate Buffer K m (mM) V max D-Ser Na + phosphate 0.87 ± 0.28 90.98 ± 13.12 K + phosphate 0.42 ± 0.08 54.56 ± 6.51 D-Thr Na + phosphate 0.45 ± 0.10 6.28 ± 1.31 K + phosphate 0.53 ± 0.09 13.83 ± 1.62 D-Allothr Na + phosphate 0.63 ± 0.08 0.40 ± 0.08 K + phosphate 0.88 ± 0.11 0.63 ± 0.09 L-Ser Na + phosphate 11.78 ± 2.53 0.32 ± 0.06 K + phosphate 10.23 ± 3.70 0.47 ± 0.11 S. R. Bharath et al. Crystal structure of D-serine deaminase FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS 2881 1.32 A ˚ . Superposition of large and small domains of StDSD with the corresponding domains of SeMetDSD results in rmsd values of 0.53 and 1.64 A ˚ , respectively. A number of local conformational changes in the small domains of WtDSD and SeMetDSD are also observed. These local changes lead to large rmsd in the compari- son of the small domains. Superposition of the Ca atoms of the large domains (Fig. 2A) in these struc- tures leaves the small domains with a residual rotation of 15°. Structures of WtDSD and SeMetDSD resemble the open and closed forms, respectively, of other Fold- type II PLP-dependent enzymes. The solvent accessible surface areas of StDSD in the open and closed forms are 16 994 and 16 540 A ˚ 2 , respectively. Ligand-induced movement of the small domain with respect to the large domain has been observed in SpSR (Fig. 2B) [23], serine racemase from Rattus norvegicus and Homo sapiens [30], and OASS from S. typhimurium [31]. In tryptophan synthase b from E. coli [32] and l-serine dehydratase from Rattus norvegicus (rat liver LSD) [9], a similar domain movement is observed between apo and holo forms of the enzymes. Consider- ing that SeMetDSD crystal was grown in the presence of the inhibitor (isoserine), it is reasonable to assume that SeMetDSD represents the ligand bound closed form of the enzyme while the WtDSD represents the unliganded open form of the enzyme. It is likely that, upon formation of external aldimine with isoserine, the enzyme undergoes conformational change to the closed Table 2. Data collection statistics. Values in parentheses refer to the highest resolution shell. R merge =(R hkl R i |I i (hkl ) ) ÆI(hkl)æ|) ⁄ R hkl RIi(hkl ), where I i (hkl) is the intensity of the ith measurement of reflection (hkl) and ÆI(hkl )æ is its mean intensity. R pim [46] = (R hkl [1 ⁄ N ) 1] 1 ⁄ 2 ) ÆI(hkl )æ|) ⁄ R hkl RI i (hkl ), where I i (hkl ) is the intensity of the ith measurement of reflection (hkl ), ÆI(hkl )æ is its mean intensity and N is the number of measurements (redundancy). I is the integrated intensity and r(I) is the estimated standard deviation of that intensity. Crystal Se-Met Se-Met Se-Met Se-Met Se-Met Wt Wavelength (A ˚ ) 0.97848 (peak) 0.97872 (inflection) 1.01876 (low remote) 0.97083 (high remote) 1.5418 (Cu Ka) 1.5418 (Cu Ka) Cell parameters (A ˚ ) a 56.26 56.37 56.41 56.38 56.46 100.02 b 187.65 187.83 187.94 187.88 188.39 46.79 c 46.48 46.53 46.54 46.57 46.59 100.04 abc 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 93.75, 90 Space group P2 1 2 1 2P2 1 2 1 2P2 1 2 1 2P2 1 2 1 2P2 1 2 1 2C2 Resolution range 45.1–2.2 (2.3–2.2) 37.6–2.0 (2.1–2.0) 41.9–2.1 (2.2–2.1) 35.3–1.8 (1.9–1.8) 48.4–1.9 (2.0–1.9) 49.9–2.4 (2.5–2.4) R merge 0.065 (0.145) 0.055 (0.226) 0.049 (0.157) 0.069 (0.304) 0.053 (0.174) 0.109 (0.492) R pim 0.023 (0.045) 0.036 (0.146) 0.028 (0.096) 0.038 (0.153) 0.019 (0.058) 0.070 (0.300) Total measurements 367 430 (52 487) 143 786 (18 234) 129 720 (16 424) 262 803 (39 256) 329 613 (45 978) 72 766 (8757) Unique reflections 25 862 (3704) 34 178 (4867) 30 465 (4311) 46 887 (6704) 36 067 (4560) 17 806 (2301) ÆI ⁄ r(I)æ 30.4 (18.4) 13.5 (4.4) 15.8 (6.0) 11.7 (3.6) 28.02 (12.0) 6.0 (2.3) Completeness 100 (100) 99.5 (98.7) 99.7 (99.0) 100 (100) 93.7 (82.5) 97.1 (87.8) Multiplicity 14.2 (14.2) 4.2 (3.7) 4.3 (3.8) 5.6 (5.9) 9.1 (10.1) 4.1 (3.8) Mosaicity 0.38 0.45 0.46 0.49 0.33 1.2 Wilson B-factor (A ˚ 2 ) 21.9 22.8 23.9 21.6 14.7 37.4 Anomalous completeness 100 (100) 90.9 (81.1) 92.7 (83.9) 99.6 (100) 91.0 (79.4) 92.9 (78.4) Anomalous multiplicity 7.6 (7.4) 2.3 (2.2) 2.3 (2.2) 2.9 (3.0) 4.9 (5.3) 2.2 (2.1) Table 3. Structure validation and refinement statistics. R work =(R hkl |F o ) F c |) ⁄ R hkl F o where F o and F c are the observed and calculated structure factors. R free [47] is calculated as for R work but from a randomly selected subset of the data (5%), which were excluded from the refinement. SeMet Native R work 0.18 0.22 R free 0.21 0.27 RMSD bond length (A ˚ ) 0.006 0.007 RMSD bond angle ( o ) 0.915 1.076 Ramachandran plot Favoured region (%) 94.4 93.1 Additionally allowed region (%) 4.7 6.3 Generously allowed region (%) 0.6 0.0 Outliers (%) 0.3 0.5 Number of Protein atoms 3307 3218 Water atoms 384 174 Non-water hetero-atoms 54 63 Average B-factor (A ˚ 2 ) Protein atoms 14.3 27.5 Water atoms 26.9 29.9 Non-water hetero-atoms 22.4 50.6 Crystal structure of D-serine deaminase S. R. Bharath et al. 2882 FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS form which is retained even after the removal of the external aldimine under crystallization conditions or from the crystals. Thus, the WtDSD and SeMetDSD structures can be viewed as open and closed forms of the enzyme. In the WtDSD structure, two segments (68–71 and 234–239) were not built due to absence of significant electron density. Of these, 68–71 stretch is involved in crystal contacts and is 27 A ˚ away from the active site. In SeMetDSD, this stretch is not involved in crystal contacts and is ordered. Therefore, the disorder observed in the WtDSD may be due to crystal pack- ing. Residues 234–239 are close to the active site and are ordered in SeMetDSD suggesting that this segment may undergo disorder–order transition upon domain closure. An aspartate residue (Asp236) occurring in this segment might have a role in catalysis (see Impli- cations for catalysis). In the closed form of the structure (SeMetDSD), an isolated electron density close to Cys276 that could correspond to an Na + ion was observed. Although Na + ions cannot be unambiguously distinguished from water molecules on the basis of electron density, bind- ing of an Na + or K + to EcDSD has been demon- strated through NMR chemical shift data [20]. Also, an equivalent site is known to bind divalent cations (Mg 2+ or Mn 2+ )inSpSR [23], serine racemase from Rattus norvegicus and Homo sapiens [30]. Therefore, an Na + ion was built into the observed density. The refined B-factor of the Na + (22.4 A ˚ 2 ) is about twice that of the atoms in its close proximity. Figure 3 shows the residues that interact with the proposed Na + ion. The charge on the Na + is neutralized by the carboxylate of Glu303. Apart from Ser307 hydroxyl and Cys309 sulfhydryl, the Na + is surrounded by three main chain carbonyl groups. A similar geometry has been observed around the bound K + in rat liver LSD [9]. In contrast, no density that could correspond to a bound ion was present at the equivalent position in WtDSD indicating that the ion binds only to the Fig. 1. Polypeptide fold of SeMetDSD showing secondary struc- tural elements. a-helices are shown as cylinders, b-strands are shown as ribbons. The two structural domains are coloured in dif- ferent shades of teal and loops are shown in red. PLP– D-Ser com- plex at the active site is shown in ball and stick representation. The Na + ion is shown as a yellow sphere. Secondary structural ele- ments which are insertions with respect to most of the other Fold- type II family of PLP-dependent enzymes are shown in orange. All secondary structural elements are labelled. Fig. 2. (A) Open and closed forms of StDSD. Large domains of WtDSD and SeMetDSD were superposed to depict the relative movement of the small domain between the two conformational states. Open conformation of WtDSD is shown in dark grey while the closed conformation of SeMetDSD is shown in light grey. The small domains are related by a residual rotation of 15°. The view is selected to highlight domain movement. (B) Large domains of open (dark grey; PDB code 1V71) and closed (light grey; PDB code 2ZPU) forms of SpSR are superposed illustrating similarity to the conformational change observed in StDSD (A). S. R. Bharath et al. Crystal structure of D-serine deaminase FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS 2883 closed form of the enzyme. Similar observation has been made in the apo form of rat liver LSD [9]. In these enzymes, the metal ion does not appear to have a catalytic role as it is not in direct contact with the substrate. Active site The active site of Foldtype II PLP-dependent enzymes is situated in a large crevice between the two domains. Based on structural comparisons with other Foldtype II PLP-dependent enzymes, PLP is expected to bind StDSD as an internal aldimine covalently bonded to the e-amino group of Lys116 situated at the beginning of helix H7. However, significant density to fit an intact PLP was not observed in the electron density maps of either WtDSD (Fig. 4A) or SeMetDSD (Fig. 4B) suggesting that there is a tendency for PLP to diffuse away from the active site. It is known that EcDSD is readily converted to the apo form when incubated with l-Cys in the presence of EDTA [15]. However, l-Cys and ⁄ or EDTA were not present in purification or crystallization steps. Also, the purified protein was yellow in colour and was catalytically active indicating that PLP is indeed bound. However, the crystals were not yellow. Enzymatic assay carried out with a dissolved SeMetDSD crystal showed low activity, which increased by a factor of 5 upon addition Fig. 3. (A) Cartoon diagram illustrating the residues of SeMetDSD that interact with Na + ion. (B) Relative position of Na + ion with respect to the modelled PLP– D-Ser complex is shown. Cys276 and Gly277, which are part of the glycine-rich loop (residues 276–282) anchoring phos- phate of the cofactor, are close to the Na + ion. Fig. 4. Electron density (2F o ) F c contoured at 1r corresponding to 0.41 electrons A ˚ )3 ) observed at the active site of (A) WtDSD and (B) SeMetDSD. The carboxyl group of modelled PLP– D-Ser is in the density corre- sponding to a bound ethylene glycol. A blob of density that represents a bound sulfate ion was observed at the site of phosphate in both the structures although density was absent for the cofactor. PLP in WtDSD and PLP– D-Ser external aldimine complex in SeMetDSD modelled by comparison with other Foldtype II enzymes are shown in ball and stick representation. Crystal structure of D-serine deaminase S. R. Bharath et al. 2884 FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS of PLP (Fig. 5A). These results suggest that the struc- tures described here most probably correspond to the apo forms. As the purified enzyme samples had cova- lently bound PLP, it is likely that the cofactor was lost during crystallization. In order to examine this possibility, 1 mgÆmL )1 WtDSD and SeMetDSD were dialysed extensively against crystallization condition. Activities of these samples with d-Ser as substrate were determined (Fig. 5B). The samples had only 20% of the activity (0.38 and 0.42 lmolÆmg )1 Æmin )1 in WtDSD and SeMetDSD, respectively) of undialysed samples suggesting that crystallization condition leads to loss of PLP. The active site pocket in both WtDSD and SeMetDSD had a blob of significant density (Fig. 4) at a position corresponding to PLP of other Foldtype II enzymes. A sulfate ion was fitted to this density as it was a component of crystallization. The sulfate ion could be refined to a B-factor (10 A ˚ 2 in SeMetDSD and 28 A ˚ 2 in WtDSD) comparable with those of the surrounding atoms (8–10 A ˚ 2 in SeMetDSD and 17–26 A ˚ 2 in WtDSD). It is worth noting that StDSD is active in the presence of excess added sulfate and hence absence of density for the PLP ring is unlikely to be due to its displacement by sulfate. As adequate density was not observed for the cofactor, PLP (internal aldimine in WtDSD) and PLP- isoserine, PLP-d-Ser, PLP-d-Thr, PLP-d-Allothr and PLP-l-Ser (external aldimines in SeMetDSD) were modelled at the active site based on the structure of rat liver LSD [9]. The close similarity in the active sites of these enzymes ensured that the modelling is reliable. A significant density was present at the active site at a position corresponding to the carboxylate of the mod- elled substrate. This density most probably corre- sponds to a bound ethylene glycol molecule. The pyridine ring of PLP occupies a cavity between Leu338 and Ile115. These residues may limit the tilting of the pyridine ring between the internal and external aldi- mine forms [31]. The side chain of Asn168 and the hydroxyl group of Thr422 are at hydrogen bonding distances from O 3 and N 1 of PLP (2.2 and 2.8 A ˚ ), respectively. Thr422 is found to be replaced by Ser (in threonine deaminase [8], O-acetylserine sulfhydrase [31] and serine racemase [23]) and Cys (in l-serine dehydra- tase [9]) in the other members of the TRPSb family of PLP-dependent enzymes. It has been proposed that Ser or Cys residue is important for maintaining the elec- tronic state of the PLP–Schiff base conjugate [33]. Pre- sumably, Thr422 fulfils the same role in StDSD. A glycine-rich loop is conserved in all the PLP-dependent enzymes and provides interactions for the binding of PLP (PLP-binding cup) [34]. This loop located at the N-terminal end of helix H12 in StDSD consists of resi- dues Gly277, Val278, Gly279, Gly280, Gly281 and Pro282. Most of these residues are conserved in all known PLP-dependent enzymes. The metal ion may stabilize the conformation of the PLP-binding loop by its interaction with carbonyl groups of Gly277 and Cys276. The carboxyl group of the substrate is held by hydrogen bonding to the Ser165 hydroxyl and amide group of Leu169. Comparison of modelled PLP-d-Ser and PLP-l-Ser complexes in SeMetDSD (Fig. 6) shows that the Ca protons of the two external aldimine com- plexes are in opposite orientations. The Ca proton in the case of PLP-d-Ser complex faces the hydroxyl group of Thr166 (2.5 A ˚ ) whereas in PLP-l-Ser com- plex it faces Lys116 (4.5 A ˚ ) and Asp236 (4.2 A ˚ ). Based on the modelled PLP–isoserine external aldimine com- plex, the hydroxyl group of Ser165 (2.7 A ˚ ) and the amide groups of Thr166 (3.3 A ˚ ) and Leu169 (2.6 A ˚ ) appear to be important for stabilizing the external aldi- mine (Fig. 7). Earlier work on EcDSD has shown that modification of a particular Cys residue leads to enzyme inactivation [15]. This might correspond to Cys276 as it is conserved and occurs near the phosphate binding loop. Fig. 5. (A) Activity of dissolved SeMetDSD crystals without (C1) and with (C2) added PLP (50 l M); 5 mMD-Ser was used as the sub- strate. (B) Activity of WtDSD and SeMetDSD under different condi- tions as described in the text. W1 and W2 correspond to WtDSD while S1 and S2 correspond to SeMetDSD. W1 and S1 correspond to holo enzyme in 50 m M HEPES pH 7.5. W2 and S2 correspond to proteins dialysed against crystallization condition. S. R. Bharath et al. Crystal structure of D-serine deaminase FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS 2885 Implications for catalysis Degradation of d-Ser to pyruvate and ammonia by StDSD involves two steps. In the first step, d-Ser is converted to aminoacrylate by Ca proton abstraction and protonation of the hydroxyl group of the substrate resulting in the release of a water molecule. In a subse- quent non-enzymatic step, aminoacrylate is converted to ammonia and the a-keto acid, pyruvate. In rat liver LSD [9], it has been observed that the N1 atom of PLP is unlikely to be protonated in view of its hydrogen bonding to the side chain S–H of Cys303. These authors also note that the PLP is likely to be in its less polarized form (HPO À 4 ) as no cation is found in its vicinity and it is held in place only by backbone amide groups of the residues from the gly- cine-rich phosphate binding loop. Based on these observations, they suggest that the Lys41 (which is linked to PLP in the internal aldimine form of the enzyme) may abstract the proton from the Ca atom of the substrate. Elimination of the substrate hydroxyl may be facilitated by the phosphate group acting as a general acid. The active site geometry of SpSR [23] is closely similar to that of rat liver LSD. Here, N1 is Fig. 6. Stereodiagram of the superposition of active sites of SeMetDSD PLP-D-Ser (light grey) and PLP-L-Ser (dark grey) complexes. The Ca proton of D-Ser points towards Thr166 in SeMetDSD and that of L-Ser points towards Lys116 and Asp236. Fig. 7. Stereodiagram of the active site geometry in WtDSD (dark grey) and SeMetDSD (light grey). PLP-D-Ser modelled in SeMetDSD is also shown. The carboxyl group of modelled PLP- D-Ser is held by hydrogen bonding with the hydroxyl group of Ser165 and the main chain amide of Leu169. Lys116 is in different orientations in these structures. Ser165 and Thr166 are closer to the substrate in SeMetDSD com- pared with WtDSD. Crystal structure of D-serine deaminase S. R. Bharath et al. 2886 FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS hydrogen bonded to the side chain of Ser308 and the phosphate is held by the amide groups from the glycine-rich phosphate binding loop. A two-base mech- anism in which Ser82 and Lys57 are involved in the abstraction of a proton from Ca of d-Ser and l-Ser, respectively, has been proposed for the racemase reac- tion. SpSR also exhibits a low level of a, b elimination of d-Ser, for which Ser82 has been proposed as the base in abstraction of the Ca proton. Examination of the active site geometry in StDSD suggests that the active site Lys116, unlike in rat liver LSD, is not at a position suitable for proton abstrac- tion (Fig. 6). Two residues, Thr166 and Tyr214, are close to Ca of the modelled external aldimine (Fig. 7) and hence might be suitable for proton abstraction. Thr166 is part of the conserved loop which holds the carboxyl group of the substrate and moves by 3.0 A ˚ when StDSD undergoes transition from the open to the closed form. The modelled SeMetDSD–PLP-d-Ser complex (Fig. 7) suggests that the Ca proton of the substrate is close to Thr166 hydroxyl (2.5 A ˚ ). Thr166 is structurally equivalent to Ser82 of SpSR. Therefore, in StDSD Thr166 may fulfil the same role. The side chain hydroxyl of Tyr214 is at a distance of 6.3 A ˚ (in SeMetDSD) from the Ca atom of the substrate and is disordered in WtDSD. It undergoes substantial dis- placement between the open and closed forms and hence may be involved in proton abstraction, although it appears to be a less likely candidate than Thr166. Further mutagenesis experiments need to be carried out to clarify the role of these residues in catalysis. As in rat liver LSD [9], PLP may be involved in the pro- tonation of the substrate hydroxyl group leading to the release of a water molecule and formation of aminoacrylate. It has been noted earlier that the amino group of the incoming amino acid should be deprotonated to make a nucleophilic attack on the C4¢ of PLP [23]. Occurrence of Tyr214 and Asp236 near the active site of StDSD suggests that these residues might be important for the initial formation of external aldimine. StDSD exhibits substantial activity with d-Thr. Mode- lling of d-Thr as an external aldimine in SeMetDSD shows no unacceptable contacts between the substrate and protein atoms. The lower rate of degradation with d-Thr may be because of a lower rate of protonation by the phosphate group. StDSD has a low level of activity with l-Ser. Modelling l-Ser at the active site suggests that the Ca proton points towards Lys116 and Asp236 and not towards Thr166 or Tyr214. Therefore, Lys166 or Asp236 may be involved in deg- radation of l-Ser by St DSD. Based on fluorescence energy transfer and CD stud- ies in EcDSD [24], it was suggested that Trp197 (equivalent to StDSD Trp195) undergoes large dis- placement during catalysis and hence could be a key residue in catalysis. However, Trp195 is not close to the active site and is unlikely to be important for the catalytic function. Conclusions StDSD is a monomeric PLP-dependent enzyme that catalyses a, b elimination of d-Ser, d-Thr, d-Allothr and l-Ser to the corresponding keto acid and ammo- nia. Structural data presented here suggest that StDSD protomer has a fold similar to those of other Foldtype II PLP-dependent enzymes and undergoes conforma- tional change from an open unliganded state to a closed liganded state. It has a low affinity for the co- factor PLP under the conditions of crystallization. An ion bound near the active site (most probably Na + ) may be essential to keep the PLP binding loop in a conformation appropriate for cofactor binding and hence for catalysis. The ion is unlikely to be directly involved in the enzyme reaction. Differences in the cat- alytic rates with respect to different substrates (Table 1) in the presence of Na + and K + suggest that these ions affect the conformation of the PLP binding loop in subtle ways. The positioning of Thr166 in these structures with respect to the substrate suggests that it is suitable for abstraction of the proton from the Ca atom of the substrate. Further structural and kinetic studies with site mutants of residues at the active site and determination of structures of ligand and inhibitor complexes will provide a deeper understanding of the catalytic mechanism of this Foldtype II PLP-dependent enzyme. Materials and methods Cloning, overexpression and purification of StDSD The dsdA gene from S. typhimurium was amplified by PCR using the following gene-specific primers: StDSD-sense pri- mer CATATGGCTAGC ATG GAA AAC ATA CAA AAG CTC ATC; StDSD-antisense primer GGATCC TTA CTCGAG GCGTCC TTT TGC CAG GTA TTG. The underlined bases correspond to restriction sites. The sense primer had NdeI and NheI restriction sites, whereas the antisense primer had BamHI and XhoI sites. The dsdA gene was cloned into pET21b between the NheI and XhoI sites. The cloning strategy was such that the expressed protein had eight extra amino acids at the C-terminus S. R. Bharath et al. Crystal structure of D-serine deaminase FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS 2887 (LEHHHHHH) that included a hexa-histidine tag. The clone obtained was confirmed by sequencing. The protein was overexpressed in E. coli BL21 (DE3) Rosetta cells. The cells were grown in LB medium containing 100 lgÆmL )1 of ampicillin at 37 °C until A 600 reached 0.5 and were then induced with 1.0 mm isopropyl thio-b-d-galactoside (IPTG) and grown at 25 °C for a further 6 h. The cells were pel- leted by centrifugation at 4810 g for 10 min and resus- pended in buffer A containing 50 mm Tris pH 8.0, 400 mm NaCl, 30% glycerol and 50 lm PLP. After sonication and centrifugation, 1 mL of Ni-nitrilotriacetic acid beads were added to 30 mL of the soluble fraction and kept for end- to-end rotation for 3 h. The unbound proteins from the column were washed using buffer B containing 50 mm Tris pH 8.0, 200 mm NaCl, 20% glycerol and 50 lm PLP. Non- specifically bound proteins were removed by a wash with buffer B containing 20 mm imidazole. In the last step, the protein was eluted using buffer B containing 200 mm imid- azole. The eluted protein was concentrated to 1 mL using centricon tubes, loaded onto a Sephacryl S-200 preparative column for a final round of purification and eluted using 50 mm Hepes buffer pH 7.5 containing 100 mm NaCl. The purified protein, free of excess PLP was concentrated to 10 mg.mL )1 in Centricon tubes and used for crystallization. The purified protein corresponded to a size of 49 kDa on a 12% SDS ⁄ PAGE. The molecular mass was confirmed by MALDI-TOF. Selenomethionine incorporation The plasmid containing dsdA gene was transformed into BL21 (DE3) pLysS strain of E. coli. The cells were grown in minimal medium. Methionine biosynthesis was inhibited by the addition of 50 mgÆL )1 of Leu, Ile, Val, Lys, Thr and Phe half an hour before induction with 1.0 mm IPTG. Sele- nomethionine was added at the time of induction. Purifica- tion of the enzyme was carried out following the same protocol as used for the native enzyme, except that all buf- fers contained 5 mm b-mercaptoethanol. The selenomethio- nine incorporation was confirmed by accurate mass determination using ESI-MS. Biochemical studies a-keto acids released from d-Ser, d-Thr, d-Allothr and l-Ser by the enzymatic action of StDSD was estimated using the 2,4-dinitrophenyl hydrazine (DNPH) method [17]. The reaction mixture for the assay with d-Ser consisted of 50 mm sodium or potassium phosphate buffer (pH 7.5), 20 lm PLP, varying concentrations of d-Ser and 50 ng of StDSD in a final volume of 50 lL. The reaction was started by the addition of d-Ser and carried out at 37 °C for 10 min. Then 50 lL of 0.1% DNPH in 2 m HCl was added to stop the reaction and the mixture was incubated at 37 °C for 2 min, followed by the addition of 150 lLof 0.4 m NaOH. After 5-min incubation at room temperature, A 540 of the resultant hydrazone was measured. The result- ing absorbance units corrected for enzyme-blank were plot- ted against substrate concentration. The assay was again carried out with d-Thr, d-Allothr and l-Ser following a similar protocol. Activity measurements were also carried out in the crystallization condition. The kinetic parameters (K m and V max ) were determined under two different condi- tions (sodium phosphate buffer pH 7.5 and potassium phosphate buffer pH 7.5). The amount of enzyme used for determining K m and V max was different with each of the substrates: 50 ng for the assay with d-Ser, 100 ng with d-Thr, and 500 ng with d-Allothr and l-Ser. The concen- trations of the substrates were varied between 0.2 and 9.5 mm. Biochemical data were analysed using graphpad prism 5 (GraphPad software Inc, La Jolla, CA, USA). The oligomeric state of the purified protein in solution was determined using an analytical gel filtration column. Crystallization and data collection Crystallization trials were carried out with Hampton Crys- tal screens 1 and 2, PEG-ion screen, and Index screens 1 and 2 using microbatch (under oil) as well as sitting-drop vapour diffusion methods. The best crystals of StDSD were obtained from 100 mm trisodium citrate, pH 6.1, containing 5mgÆmL )1 StDSD, 0.8 m lithium sulfate and 0.4 m ammo- nium sulfate in the hanging-drop vapour diffusion method. Prior to crystallization, SeMetDSD (10 mgÆmL )1 )in50mm Hepes buffer pH 7.5, 100 mm NaCl, was incubated with 40 mmdl-isoserine for about an hour at 4 °C and n-octyl- b-glucopyranoside was added to 0.1%. Using this sample, crystals were obtained under the same condition as that of the WtDSD crystals. The quality of these crystals was bet- ter than the WtDSD crystals or WtDSD crystals obtained in the presence of isoserine. The crystals were mounted on a cryo-loop and frozen in liquid nitrogen for X-ray diffraction data collection. Data on a WtDSD crystal were collected to 2.4 A ˚ resolution using a Bruker AXS Microstar rotating anode X-ray generator and a MARRESEARCH image plate detector system. The data were processed using mosflm and scala of the CCP4 suite [35]. These crystals belonged to the space group C2 with unit cell parameters a = 100.02 A ˚ , b = 46.80 A ˚ , c = 100.04 A ˚ and b = 93.75°. The crystal asymmetric unit was compatible with a monomer (solvent content 47.9%). Data on SeMetDSD crystals were collected at four different wavelengths, namely 0.97848 A ˚ (peak), 0.97872 A ˚ (inflection point), 0.97083 A ˚ (high energy remote) and 1.01876 A ˚ (low energy remote) at beamline 14 of ESRF, Grenoble, France. The best of these data sets extended to 1.8 A ˚ resolution. The data were processed using mosflm and scala of the CCP4 suite. The crystals belonged to the orthorhombic space group P2 1 2 1 2 with a = 56.4 A ˚ , b = 188.4 A ˚ and c = 46.59 A ˚ . The asymmetric unit was compatible Crystal structure of D-serine deaminase S. R. Bharath et al. 2888 FEBS Journal 278 (2011) 2879–2891 ª 2011 The Authors Journal compilation ª 2011 FEBS [...]... description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families BMC Bioinformatics 10, 273 7 Gallagher DT, Gilliland GL, Xiao G, Zondlo J, Fisher KE, Chinchilla D & Eisenstein E (1998) Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase Structure 6, 46 5–4 75 8 Simanshu DK, Savithri HS & Murthy MR (2006) Crystal structures. .. 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Crystal structure of D-serine deaminase Department of Science and Technology (DST) and the Department of Biotechnology (DBT), Government of India The other four data sets were collected at BM14 of ESRF, Grenoble, France We thank the staff at the X-ray laboratory in MBU and the beam line scientists at BM14, ESRF, for their cooperation MRNM and HSS thank the DST and DBT for funding SRB and SB acknowledge . Crystal structures of open and closed forms of D-serine deaminase from Salmonella typhimurium – implications on substrate specificity and catalysis Sakshibeedu Rajegowda. enzymes and undergoes conforma- tional change from an open unliganded state to a closed liganded state. It has a low affinity for the co- factor PLP under the conditions of crystallization. An ion. P2 1 2 1 2P2 1 2 1 2P2 1 2 1 2P2 1 2 1 2P2 1 2 1 2C2 Resolution range 45. 1–2 .2 (2. 3–2 .2) 37. 6–2 .0 (2. 1–2 .0) 41. 9–2 .1 (2. 2–2 .1) 35. 3–1 .8 (1. 9–1 .8) 48. 4–1 .9 (2. 0–1 .9) 49. 9–2 .4 (2. 5–2 .4) R merge 0.065 (0.145) 0.055 (0.226)