JOURNAL OF BACTERIOLOGY, Sept. 2009, p. 5832–5837 0021-9193/09/$08.00⫹0 doi:10.1128/JB.00599-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 18
Crystal Structures of Streptococcus suis Mannonate Dehydratase (ManD) and Its Complex with Substrate: Genetic and Biochemical Evidence for a Catalytic Mechanism䌤† Qiangmin Zhang,1,2 Feng Gao,1 Hao Peng,1 Hao Cheng,1,2 Yiwei Liu,1 Jiaqi Tang,3 John Thompson,4 Guohua Wei,5 Jingren Zhang,6 Yuguo Du,5 Jinghua Yan,1 and George F. Gao1,2,7* CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China1; Graduate University, Chinese Academy of Sciences, Beijing 100049, China2; Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing 210002, China3; Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 208924; Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China5; Center For Immunology and Microbial Disease, Albany Medical College, Albany, New York6; and Beijing Institutes of Life Science, Chinese Academy of Sciences, Lincui East Road, Beijing 100101, China7 Received 7 May 2009/Accepted 12 July 2009
Mannonate dehydratase (ManD) is found only in certain bacterial species, where it participates in the dissimilation of glucuronate. ManD catalyzes the dehydration of D-mannonate to yield 2-keto-3-deoxygluconate (2-KDG), the carbon and energy source for growth. Selective inactivation of ManD by drug targeting is of therapeutic interest in the treatment of human Streptococcus suis infections. Here, we report the overexpression, purification, functional characterization, and crystallographic structure of ManD from S. suis. Importantly, by Fourier transform mass spectrometry, we show that 2-KDG is formed when the chemically synthesized substrate (D-mannonate) is incubated with ManD. Inductively coupled plasma-mass spectrometry revealed the presence of Mn2ⴙ in the purified protein, and in the solution state catalytically active ManD exists as a homodimer of two 41-kDa subunits. The crystal structures of S. suis ManD in native form and in complex with its substrate and Mn2ⴙ ion have been solved at a resolution of 2.9 Å. The core structure of S. suis ManD is a TIM barrel similar to that of other members of the xylose isomerase-like superfamily. Structural analyses and comparative amino acid sequence alignments provide evidence for the importance of His311 and Tyr325 in ManD activity. The results of site-directed mutagenesis confirmed the functional role(s) of these residues in the dehydration reaction and a plausible mechanism for the ManD-catalyzed reaction is proposed. The crystal structures of ManDs from Enterococcus faecalis and from two archaeal species (Chromohalobacter salexigens and N. aromaticivorans) have been solved (Protein Data Bank [PDB] codes 1TZ9, 3BSM, and 2QJJ, respectively), but a detailed structural description of E. faecalis ManD has not yet been published. E. faecalis ManD was structurally assigned to the xylose isomerase-like superfamily, whose members contain a canonical TIM barrel, (␣)8. This structure is characterized by eight parallel -strands, forming a central -barrel, and eight surrounding ␣-helices that alternate along the peptide backbone. The monomer of homotetrameric ManD from N. aromaticivorans contains two domains comprising an N-terminal ␣⫹ capping domain and a C-terminal modified TIM barrel, (␣)7, as is the case of L-rhamnonate dehydratase (RhamD) from E. coli (17). Based on the crystal structure and mutational analysis, catalytic roles have been proposed for residue Tyr159 or His212 in ManD from N. aromaticivorans (18). It is noteworthy that ManDs from both C. salexigens and N. aromaticivorans are members of the mandelate racemaselike subfamily of the enolase superfamily. In the present study, ManD from S. suis was expressed, purified, and functionally characterized, and the crystal structures of ManD in both native and substrate Mn2⫹-bound forms were solved. Analogous to the E. faecalis enzyme, ManD from S. suis is homodimeric, in contrast to the two archaeal ManDs
Streptococcus suis serotype 2 is an emerging zoonotic pathogen that is of some concern to public health, particularly in the light of the recent emergence of a new disease form of streptococcal toxic shock syndrome (22, 25). Some microorganisms, including S. suis, can metabolize glucuronate via the EntnerDoudoroff pathway as the sole carbon and energy source for growth (5, 12, 26). Mannonate dehydratase (ManD; EC 4.2.1.8, also known as mannonate hydratase) is encoded by the gene uxuA in S. suis (3) and is the third enzyme in the pathway for dissimilation of glucuronate to 2-keto-3-deoxygluconate (2KDG). In this third step, ManD catalyzes the removal of a molecule of water from D-mannonate to yield 2-KDG that is subsequently converted to glyceraldehyde 3-phosphate and pyruvic acid (14, 20). To date, biochemical properties and physiological functions have been reported for ManDs from several species including Escherichia coli (5, 19), Bacillus subtilis (14), Bacillus stearothermophilus (20), and Novosphingobium aromaticivorans (18). * Corresponding author. Mailing address: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing 100101, People’s Republic of China. Phone: 86 10 64807688. Fax: 86 10 64807882. E-mail:
[email protected]. † Supplemental material for this article may be found at http://jb .asm.org/. 䌤 Published ahead of print on 17 July 2009. 5832
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that exist as homotetramers in the solution state. Structural analyses and residue alignments permitted tentative identification of catalytically important residues. The results of site-directed mutagenesis experiments confirmed the functional roles of these residues. Based on our findings and on the structure of ManD from E. faecalis, a plausible dehydration mechanism is proposed for the ManD-catalyzed reaction.
MATERIALS AND METHODS Protein expression and purification. The uxuA gene coding for S. suis serotype 2 mannonate dehydratase (ManD) was cloned by using PCR with genomic DNA (strain 05ZYH33) as a template and the primers 5⬘-GGAATTCCATATGAAA ATGTCATTTCGCTG-3⬘ and 5⬘-CCGCTCGAGTTATCCCTCCTTTGTTCC3⬘. The PCR product was digested with the restriction endonucleases NdeI and XhoI and cloned into the prokaryotic expression vector pET28b(⫹) (Novagen) to generate the plasmid pET28b-uxuA. The identity of the DNA construct with an N-terminal His6 tag was verified by DNA sequencing. E. coli BL21(DE3) cells transformed with the appropriate plasmid were grown in LB medium containing 50 g ml⫺1 kanamycin at 37°C until an optical density at 600 nm of 0.6 to 0.8 was reached. Expression of ManD was induced by the addition of 0.1 mM isopropyl-D-thiogalactopyranoside to the culture, and organisms were grown at 37°C for 4 h. The induced cells were harvested by centrifugation and resuspended in a lysis buffer containing 20 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 10 mM imidazole, 0.1% Triton X-100, and 100 M phenylmethylsulfonyl fluoride. The cells were lysed by sonication (on ice), and the resulting supernatants were loaded onto an Ninitrilotriacetic acid affinity column (Qiagen) equilibrated with a solution containing 20 mM Tris-HCl (pH 8.0), 0.2 M NaCl, and 10 mM imidazole. The protein was eluted with an increasing linear concentration gradient of imidazole and then dialyzed against 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl. After concentration, the preparation was loaded onto a Superdex 200 fast protein liquid chromatography column (Amersham Biosciences) equilibrated with the same dialysis buffer. Fractions containing ManD were pooled and concentrated in an Amicon Ultra-15 filter (Millipore) using a 10-kDa cutoff membrane. Protein purity was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the native molecular weight of ManD was estimated by nondenaturing native-PAGE. The ManD mutant proteins H311A and Y325F were constructed utilizing a QuikChange site-directed mutagenesis kit (Stratagene). Proteins were purified as described above for parental ManD. Protein concentrations were determined using a bicinchoninic protein assay according to the manufacturer’s instructions (Pierce). Crystallization. Purified ManD was concentrated to 10 mg ml⫺1 in a buffer containing 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl. The protein was crystallized using hanging-drop vapor diffusion at 291 K by mixing 1 l of protein solution with 1 l of reservoir solution containing 0.2 M potassium-sodium tartrate, 0.1 M sodium citrate (pH 6.5), and 1 M ammonium sulfate. Crystals of the native enzyme, with excellent diffraction, appeared within 3 to 7 days. Crystals of ManD in complex with D-mannonate were prepared by the same hangingdrop procedure at 277 K, but in this case the reservoir contained 0.2 M potassium sodium tartrate tetrahydrate, 0.1 M tri-sodium citrate dehydrate (pH 6.5), 1 M ammonium sulfate, and 15 mM D-mannonate. Data collection, structure determination, and refinement. For data collection, crystals were cryoprotected by being soaked for 3 min in the reservoir solution supplemented with 15% glycerol and then rapidly cooled to 100 K in a stream of gaseous nitrogen. X-ray diffraction images were collected on an R-axis image plate mounted on a Rigaku rotating copper anode. The data for the native protein crystal were processed using the programs MOSFLM (11) and SCALA (7). The data for the complex crystal were indexed, processed, and scaled using HKL2000 (16). The structure of native ManD was solved by molecular replacement using the program MolRep (23) with the E. faecalis ManD as a search model (PDB code 1TZ9). The structure was rebuilt and refined using COOT (6) and the REFMAC5 program (15). The native enzyme crystal belongs to the tetragonal space group P43212 (a ⫽ b ⫽ 105.7 Å, c ⫽ 159.8 Å, and ␣ ⫽  ⫽ ␥ ⫽ 90°) with two molecules in the asymmetric unit. The final refined model from the native enzyme data set was used as the initial model for the determination of the structure of the enzyme complex. REFMAC5 (15) was again used in conjunction with COOT (6) to refine and build the complex crystal structure. Ligands were fitted to the difference electron density, and further rounds of restrained refinement were carried out. Identification of sites of solvent molecules was performed by the automatic water-picking algorithm of COOT (6). The positions of these automatically picked water molecules were manually checked, and a few more
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TABLE 1. Data collection and refinement statistics Value for the indicated ManD structurea Parameter Native
Data collection Space group Unit cell (Å) Resolution range (Å) No. of unique reflections Completeness (%) Rmergeb Avg I/ (I) Refinement Resolution range (Å) Rworkc Rfreed RMS bond (Å) RMS angle (°) No. of protein atoms No. of water molecules Bound ligand(s) No. of ligand atoms
Complex
P43212 P43212 a ⫽ 105.7, b ⫽ 105.7, a ⫽ 105.5, b ⫽ 105.5, c ⫽ 159.8 c ⫽ 160.1 50-2.9 50-2.9 20,728
20,245
100 0.12 (0.37) 11.8 (5.7)
97 0.04 (0.50) 44.0 (6.9)
105.4-2.9
88.0-2.9
0.23 0.28 0.014 1.6 5,473
0.23 0.28 0.013 1.6 5,469
26
46
Mn2⫹ 1
D-mannonate,
Mn2⫹
15
a
The values in parentheses are those for the highest-resolution shell. Rmerge ⫽ ⌺兩I(k) ⫺ 关I兴 兩/⌺I(k), where I(k) is the value of the kth measurement of the intensity of reflection, and 关I兴 is the mean of the intensity of that reflection. c Rwork ⫽ ⌺兩Fo ⫺ Fc兩/⌺Fo for the 95% of the reflection data used in the refinement. Fo and Fc are the observed and calculated structure factor amplitudes, respectively. d Rfree is the equivalent of Rwork, except that it was calculated for a randomly chosen 5% test set excluded from the refinement. b
water molecules were manually identified on the basis of electron density contoured at 1.0 in the 2Fo ⫺ Fc map and 3.0 in the Fo ⫺ Fc map (where Fo is the observed and Fc is the calculated structure factor amplitude). The data collection and final refinement statistics are given in Table 1. For the structures of the S. suis ManD in native and complexed forms, the whole polypeptide is well defined in the electron density map (see Fig. S1 in the supplemental material). However, some surface residues (140 to 155) are disordered and were not included in the two structures. A clear electron density of D-mannonate in the A molecule was observed in the ManD complex structure. However, the electron density of D-mannonate in the B molecule was disordered and excluded in the final structure. The Mn2⫹ ions are clearly visible in the active sites of the native and complexed structures of ManD. Final models were validated using PROCHECK (9). Structure figures were generated by PyMOL (4), unless otherwise noted. Metal ion analysis. Purified ManD protein was concentrated, and equal volumes of protein solution and flowthrough (after fast protein liquid chromatography) as the control were treated with 1 M HNO3. Metal ion analysis by inductively coupled plasma-mass spectrometry (ICP-MS) was carried out at the Analysis Centre of Tsinghua University, Beijing, China. Synthesis of D-mannonate and detection of reaction product by FTMS. Dehydration of D-mannonate by ManD was determined by Fourier transform mass spectrometry (FTMS). The sample for FTMS analysis (400 l at 25°C) contained 8 mM D-mannonate (see Fig. S2 in the supplemental material), 200 mM TrisHCl, pH 7.5, 8 mM MnSO4, and 1 M ManD. The mixture was incubated for 3 h at 37°C, and the reaction was stopped by the addition of 600 l of 10% trichloroacetic acid. Precipitate was removed by centrifugation, and the clarified supernatant was subjected to FTMS analysis. A similarly prepared solution containing D-mannonate (no enzyme) served as the control. Kinetic characterization of mutant and native ManD. The mutant and wildtype ManD activities were assayed according to a published procedure (19). Briefly, ManD activity (at 37°C) was followed spectrophotometrically by monitoring the formation of the complex between thiobarbituric acid and the reaction product 2-KDG. The assay mixture contained increasing concentrations of
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D-mannonate, 200 mM Tris-HCl buffer (pH 7.5), 8 mM MnSO4, and 1 M ManD. For wild-type ManD, an additional assay at 4°C was also performed. The initial velocity versus substrate concentration was analyzed by direct fitting of data points to the Michaelis-Menten equation using the enzyme kinetics module from SigmaPlot software. Protein structure accession number. The coordinates and structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank under accession codes 3FVM (native ManD) and 3DBN (complexed ManD).
RESULTS Identification of divalent cation in native ManD by ICP-MS. The magenta color of the purified enzyme in solution indicated the presence of metal ion(s) bound to ManD. Data obtained by ICP-MS showed that the protein sample (15 mg ml⫺1) contained the following: 18 g ml⫺1 Mn2⫹, 0.3 g ml⫺1 Mg2⫹, 4 g ml⫺1 Zn2⫹, and 4.5 g ml⫺1 Ni2⫹ (presumably from the Ni-nitrilotriacetic acid affinity purification step). The color of the protein solution implies that this protein might bind Mn2⫹ ion, and the calculated molar ratio of Mn2⫹ ion to each ManD monomer is about 1:0.9, indicating that each ManD monomer roughly has one Mn2⫹ ion-binding site theoretically. It is important to note that no divalent metal ions were included in any solutions used during purification of the protein. The ICP-MS data provided support for Mn2⫹ ion as a likely component of the active site of ManD, as suggested by Dreyer (5). Identification of ManD-catalyzed reaction product by FTMS. The protein (YP_001198523.1) encoded by the uxuA gene of S. suis is annotated bioinformatically as ManD. The putative substrate for this enzyme (D-mannonate) is not commercially available, and, accordingly, a small quantity of this compound was chemically synthesized for characterization of the enzyme. The availability of this compound enabled us to unequivocally determine the product of ManD catalysis by FTMS analysis. As shown in Fig. 1A, the spectrum of an enzyme-free reaction mixture revealed a single dominant peak at m/z 197.06207, consistent with the molecular weight of protonated D-mannonate. By contrast, after incubation with ManD, two dominant peaks were found. The first peak at m/z 197.06607 is clearly attributable to remaining substrate, whereas the m/z 179.02881 of the second peak is consistent with formation of the expected protonated dehydration product, 2-KDG. Oligomeric state of ManD. The results of gel filtration, SDSPAGE, and native-PAGE suggest that ManD from S. suis exists as a homodimer in solution and thus differs from the tetrameric form assumed by ManD of N. aromaticivorans (18). As illustrated in Fig. 1B, denatured ManD from S. suis exhibited a single band with a molecular mass of ⬃41 kDa by SDS-PAGE. However, the results of native-PAGE indicated a molecular mass of between 66 to 132 kDa, and during gel filtration ManD eluted at the volume expected for a protein with a mass of ⬃80 kDa (Fig. 1B). Structure of ManD. The polypeptide chains of homodimeric S. suis ManD are related to each other by a twofold axis. The overall surface area buried in the dimeric interface is about 3,800 Å2 per subunit. The interface is maintained by numerous hydrogen bonds formed between side chain functional groups from one subunit and backbone atoms from another subunit or between side chain groups contributed by each monomer. In addition, the side chains of several hydrophobic residues
FIG. 1. (A) FTMS from the electrospray ionization of the product and remaining substrate during the dehydration reaction. (Top) Highresolution electrospray ionization-FTMS spectrum of the protonated D-mannonate ion. (Bottom) High-resolution electrospray ionizationFTMS spectrum of the reaction mixture with ManD showing the dehydrated product ion (left) and remaining protonated D-mannonate ion (right), respectively. (B) Protein purification. SDS-PAGE (left inset), nondenaturing PAGE (right inset), and gel filtration chromatograms. The lane numbers of the SDS-PAGE sample correspond to the numbers in the elution profile. In the nondenaturing PAGE diagram, lane a shows the purified ManD obtained by gel filtration; lanes b and c contain molecular mass markers. OD, optical density; AU, arbitrary units.
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FIG. 2. Cartoon diagram of the S. suis ManD monomer. The core structure of S. suis ManD is a TIM (␣)8 barrel. The connecting ␣-helices and -sheet composed of four -strands are shown in green and light blue, respectively. The Mn2⫹ ion is shown as an orange sphere. In the monomer structure of the complex, the substrate is close to metal ion and is depicted by yellow sticks.
(including Pro23, Leu282, Trp316, Leu332, and Ile356) project into the dimeric interface. Each monomer has a globular shape with one long protruding C-terminal arm (Fig. 2). The polypeptide chain of ManD contains 366 amino acid residues. The core structure of the ManD monomer is a TIM barrel (2) comprising eight ␣-helices (␣1 to ␣8) and a -barrel composed of eight parallel -strands (1 to 8). Instead of the connecting loops found in the canonical TIM barrel, part of the secondary structural elements of the TIM barrel in the ManD structure are connected by an additional five ␣-helices (␣3⬘, ␣4⬘, ␣4⬙, ␣6⬘, and ␣7⬘) and four -strands (4⬘, 4⬙, 7⬘, and 7⬙). On top of the TIM barrel, four additional -strands (4⬘, 4⬙, 7⬘, and 7⬙) form an antiparallel -sheet that is exposed to the aqueous environment. There is also a -turn between the ␣5-helix and 6-strand. A divalent metal ion-binding site is located in the opening of the TIM barrel, and in the complex structure D-mannonate is adjacent to the Mn2⫹ ion. Comparison with ManD proteins from other species. Although present in more than 100 bacterial species, ManD has not yet been found in humans. Comparative alignment of amino acid sequences (see Fig. S3 in the supplemental material) reveals that S. suis ManD has 26 to 40.8% sequence identity with other ManDs from several species of eubacteria, including E. faecalis, Thermotoga maritima, Flavobacteria bacterium BBFL7, Salmonella choleraesuis, E. coli K12, Moorella thermoacetica strain ATCC 39073, S. suis 89/1591, B. subtilis, and Thermoplasma acidophilum. The strictly conserved residues surrounding the substrate at the active site include Glu64, Trp111, His199, Asp202, His266, Arg268, Arg308, Asp310, His311, Pro323, and Tyr325. And the other two residues, Arg6 and Tyr8, surrounding the substrate are highly conserved (among 9 of the 10 aligned sequences). A search of the Pfam database (21) shows that ManD from S. suis structurally belongs to the xylose isomerase-like superfamily, in which a portion of the connecting loops of the canonical TIM barrel are replaced by secondary structural elements. The structure of the S. suis ManD closely resembles that of ManD from E. faecalis (PDB
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code 1TZ9) (unpublished data), with 40.8% sequence identity and 1.0 Å of root mean square (RMS) deviation over 294 equivalent C␣s (see Fig. S4 in the supplemental material). Recently, two ManD structures from the archaeal species C. salexigens (PDB code 3BSM) (unpublished data) and N. aromaticivorans (PDB code 2QJJ) (18) have been determined. These two proteins are members of mandelate racemase-like subfamily of the enolase superfamily, and ManD from S. suis displays a comparatively low level of sequence identity with the ManD proteins from C. salexigens and N. aromaticivorans (12% and 12.6%, respectively). Proteins of the mandelate racemaselike subfamily have an N-terminal ␣⫹ capping domain and a modified TIM barrel C-terminal domain (only seven ␣-helices), whereas the structure of S. suis ManD is a canonical TIM barrel with nine secondary structural elements involving in the (␣)8 connection. The complex structure and active site architecture of ManD. The overall structure of the complexed ManD is similar to that of its native form with a very low RMS deviation of 0.3 Å for 663 ␣-carbons. The D-mannonate substrate is well defined in the final electron density map of the ManD complex structure (Fig. 3A). Residues Arg6, Tyr8, Trp111, His199, Arg308, Asp310, His311, Pro323, and Tyr325 form a substrate “pocket” in the TIM barrel at distances less than 3.5 Å from the substrate. Residues with the potential to form hydrogen bonds (2.4 to 3.4 Å) with the substrate include Arg6, Tyr8, Trp111, Asp202, and Tyr325. The two amino acids, His311 and Tyr325, are favorably positioned within the complex structure for catalysis to occur. His311 is positioned within 3.2 Å of C-2-H position, and Tyr325 is within hydrogen-bonding distance (2.4 Å) to the hydroxyl group at C-3 of the substrate. As observed in the structures of other acid sugar dehydratases (18, 24), the divalent Mn2⫹ ion in the complex structure participates in a sixfold coordination with the side chain oxygen atom of Asp310, the thiol group of Cys237, the carboxyl oxygen atom and C-2-OH group of the substrate, and the nitrogen atoms of His199 and His266 (Fig. 3A). Since two oxygen-containing groups of the substrate are involved in Mn2⫹ coordination, this metal ion presumably plays an important role in the recognition and orientation of D-mannonate within the active site. In the native ManD structure, all the metal liganding side chains of the protein are present and interact with the Mn2⫹ in a fashion identical to the case when the substrate mannonate is present. A water molecule is found in close contact with the Mn2⫹ ion instead of the substrate, as shown in Fig. 3B. Site-directed mutagenesis of catalytic residues. From the previous discussion, we concluded that His311 and Tyr325 were functionally important residues. To obtain experimental support for our hypothesis, site-directed mutagenesis of the two residues was carried out to yield the mutant proteins H311A and Y325F. The proteins were highly expressed and purified, and their enzymatic activities were determined (Table 2). Wild-type ManD shows a relatively high affinity and catalytic efficiency for D-mannonate (Km ⫽ 3 ⫾ 0.19 mM and kcat ⫽ 5.88 s⫺1, respectively). In marked contrast, the mutant protein Y325F was catalytically inactive while the activity of H311A was only 2.4% that of the native enzyme. Fortuitously, ManD exhibited no enzymatic activity at 4°C, and this finding facilitated crystallization of the native protein in complex with its intact substrate.
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FIG. 3. (A) Stereoview of the electron density map around the substrate D-mannonate (cyan stick) and metal ion Mn2⫹ (magenta sphere) in the active site of ManD. The 2 Fo ⫺ Fc map is contoured at 1. The residues forming the substrate pocket are labeled, shown in ball-and-stick models, and colored by atom. The hydrogen bonding interactions between the residues and substrate are shown as dotted red lines. The coordination interaction of metal ion is represented as dotted black lines. (B) The coordination geometry of the Mn2⫹ (magenta sphere) ion in the native ManD structure. One water molecule (Wat; red sphere) is found in close contact to this ion instead of the substrate of the complex structure.
DISCUSSION FTMS analysis of the reaction mixture established that the protein, designated YP_001198523.1, has ManD activity and can catalyze the dehydration of D-mannonate to yield 2-KDG as the reaction product at 37°C. But no detectable activity of ManD was observed at 4°C, which is exactly the condition where the ManD complex crystal is obtained. Generally speaking, both the substrate and product of ManD could be bound at the active site; the fact that the substrate predominates in the difference density map of the complex structure (Fig. 3A), favorable to the elucidation of the ManD mechanism, is consistent with the enzymatic data at 4°C mentioned above. Certainly, we cannot entirely exclude the possible existence of the trivial product in the active site. Sequence alignment of ManDs from eubacterial species and structural analysis of S. suis ManD itself suggested important
TABLE 2. Kinetic parameters for D-mannonate dehydratase, wild-type and mutant enzymes, measured at pH 7.5 Protein
Temp (°C)
Km (mM)
kcat (s⫺1)
kcat/Km (mM⫺1 䡠 s⫺1)
Wide type H311A Y325F Wide type
37 37 37 4
3.0 ⫾ 0.19 1.9 ⫾ 0.13 Inactive Inactive
5.88 0.09
1.96 0.047
roles for His311 and Tyr325 in ManD-catalyzed dehydration. The results of kinetic analyses (Table 2) with mutant forms of S. suis ManD provided strong evidence to support our hypothesis. Indeed, basic residues frequently participate in the catalytic process of many dehydratases (8, 10, 13, 24), and tyrosine has also been found to play a critical role in dehydration reactions (1, 18). Based on functional assays, comparative sequence alignments, structural analysis, and the results of mutagenesis, a mechanism for the ManD-catalyzed reaction in S. suis is proposed (Fig. 4). In this scheme, the dehydration process begins with abstraction of the C-2 proton by His311 and formation of an enediolate intermediate that is stabilized by the divalent Mn2⫹ ion. Subsequently, Tyr325 facilitates the acid-catalyzed departure of the C-3-OH group to yield one water molecule and an enol intermediate that undergoes stereospecific ketonization to 2-KDG, the reaction product. The dehydration mechanism advanced in Fig. 4 is compatible with our structure and enzyme kinetics data. As anticipated, the site-directed mutation Y325F resulted in the loss of all ManD activity. Site-directed mutation of the equally essential histidine residue (H311A) also caused the loss of ⬎97% of ManD activity. The reasons for this remaining low level of activity are not entirely clear, but one might suggest that the proton acceptor role of His311 is partially replaced by environmental radical OH⫺. This mechanism is somewhat analogous to that suggested for ManD from N. aromaticivorans (18). The divalent cation (Mn2⫹ in S. suis ManD; Mg2⫹ in N. aromaticivorans
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FIG. 4. Proposed dehydration mechanism catalyzed by S. suis ManD. The C-2 proton of the substrate is presented to, and extracted by, the basic His311 residue. Tyr325 promotes the acid-catalyzed departure of the C-3-OH group, and the Mn2⫹ ion stabilizes the enolate intermediate that is formed during the dehydration reaction.
ManD) stabilizes the enediolate intermediate. But for the proposed mechanism of the latter dehydratase, the deprotonation role was assigned to Tyr159 (analogous to H311 in case of S. suis ManD), and the acid-catalyzed departure of the C-3-OH group may be catalyzed by Tyr159 and/or His212 (analogous to Tyr325 in case of S. suis ManD). Notably, our scheme emphasizes the independent, definite role(s) of the two key residues (histidine and tyrosine) in the reaction catalyzed by ManD from S. suis. ACKNOWLEDGMENTS We acknowledge the assistance of the staff in the Analysis Centre of Tsinghua University in metal ion analysis. We are grateful to Abdul Hamid Khan, Jie Yin, and Jianxun Qi for discussion and data collection. This work was supported by grants from the Ministry of Science and Technology (Project 973, grant 2005CB523001; National Key Technology R&D Program 2006BAD06A04; International Collaborative Projects 2007DFC30240 and 2006DFB32010) and the National Natural Science Foundation of China (grants 30770024 and 30728014). J.T. was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892. G.F.G is a Distinguished Young Investigator of the NSFC (grant no. 30525010). REFERENCES 1. Allard, S. T., W. W. Cleland, and H. M. Holden. 2004. High resolution X-ray structure of dTDP-glucose 4,6-dehydratase from Streptomyces venezuelae. J. Biol. Chem. 279:2211–2220. 2. Banner, D. W., A. C. Bloomer, G. A. Petsko, D. C. Phillips, C. I. Pogson, I. A. Wilson, P. H. Corran, A. J. Furth, J. D. Milman, R. E. Offord, J. D. Priddle, and S. G. Waley. 1975. Structure of chicken muscle triose phosphate isomerase determined crystallographically at 2.5 Å resolution using amino acid sequence data. Nature 255:609–614. 3. Chen, C., J. Tang, W. Dong, C. Wang, Y. Feng, J. Wang, F. Zheng, X. Pan, D. Liu, M. Li, Y. Song, X. Zhu, H. Sun, T. Feng, Z. Guo, A. Ju, J. Ge, Y. Dong, W. Sun, Y. Jiang, J. Wang, J. Yan, H. Yang, X. Wang, G. F. Gao, R. Yang, J. Wang, and J. Yu. 2007. A glimpse of streptococcal toxic shock syndrome from comparative genomics of S. suis 2 Chinese isolates. PLoS ONE 2:e315. 4. DeLano, W. L. 2002. The PyMOL molecular graphics system. DeLano Scientific, Palo Alto, CA. 5. Dreyer, J. L. 1987. The role of iron in the activation of mannonic and altronic acid hydratases, two Fe-requiring hydro-lyases. Eur. J. Biochem. 166:623– 630. 6. Emsley, P., and K. Cowtan. 2004. COOT: model-building tools for molecular graphics. Acta Crystallogr. D 60:2126–2132. 7. Evans, P. R. 1997. SCALA. Joint CCP4/ESF-EACBM Newsl. Protein Crystallogr. 33:22–24. 8. Koster, S., G. Stier, R. Ficner, M. Holzer, H. C. Curtius, D. Suck, and S. Ghisla. 1996. Location of the active site and proposed catalytic mechanism of pterin-4a-carbinolamine dehydratase. Eur. J. Biochem. 241:858–864. 9. Laskowski, R. A., M. W. MacArthur, D. S. Moss, and J. M. Thornton. 1993. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26:283–291.
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