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Originally published In Press as doi:10.1074/jbc.M607340200 on December 13, 2006

J. Biol. Chem., Vol. 282, Issue 8, 5770-5780, February 23, 2007
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Structural and Mutational Analyses of Drp35 from Staphylococcus aureus

A POSSIBLE MECHANISM FOR ITS LACTONASE ACTIVITY*

Yoshikazu Tanaka{ddagger}§, Kazuya Morikawa, Yu Ohki, Min Yao{ddagger}, Kouhei Tsumoto§, Nobuhisa Watanabe{ddagger}, Toshiko Ohta, and Isao Tanaka{ddagger}1

From the {ddagger}Faculty of Advanced Life Sciences, Hokkaido University, Sapporo 060-0810, Japan, the §Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Tokyo 277-8562, Japan, and the Department of Microbiology, Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8575, Japan

Received for publication, August 2, 2006 , and in revised form, November 30, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Drp35 is a protein induced by cell wall-affecting antibiotics or detergents; it possesses calcium-dependent lactonase activity. To determine the molecular basis of the lactonase activity, we first solved the crystal structures of Drp35 with and without Ca2+; these showed that the molecule has a six-bladed beta-propeller structure with two calcium ions bound at the center of the beta-propeller and surface region. Mutational analyses of evolutionarily conserved residues revealed that the central calcium-binding site is essential for the enzymatic activity of Drp35. Substitution of some other amino acid residues for the calcium-binding residues demonstrated the critical contributions of Glu48, Asp138, and Asp236 to the enzymatic activity. Differential scanning calorimetric analysis revealed that the loss of activity of E48Q and D236N, but not D138N, was attributed to their inability to hold the calcium ion. Further structural analysis of the D138N mutant indicates that it lacks a water molecule bound to the calcium ion rather than the calcium ion itself. Based on these observations and structural information, a possible catalytic mechanism in which the calcium ion and its binding residues play direct roles was proposed for the lactonase activity of Drp35.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Staphylococcus aureus is a major cause of hospital- and community-acquired infections. S. aureus causes serious and fatal diseases, such as toxic shock syndrome or septicemia (1). Moreover, S. aureus has the remarkable and unfortunate feature that it can become readily resistant to antibiotics. Indeed, it has acquired resistance to almost all antibiotics so far, resulting in an increase in incidence of acute hospital-acquired infections (2).

Extensive studies have focused on how S. aureus acquires resistance to antibiotics, and genome sequencing analysis confirmed the existence of many resistance genes acquired by horizontal transfer from other species (3). In addition, S. aureus can cope with antibiotic stresses in an adaptive manner through regulation of the expression of many genes (4).

Drp35 (a 35-kDa drug-responsive protein) is a cytoplasmic protein originally found to be markedly induced upon exposure of S. aureus to cell wall-affecting antibiotics (5). Antibiotic susceptibility experiments using a drp35 defective strain and overexpressing strain of S. aureus revealed that Drp35 is correlated with bacitracin resistance, although it did not show significant changes in minimal inhibitory concentration for beta-lactams, glycopeptides, or fosfomycin (6). Drp35 can also be induced by a variety of detergents, including Nonidet P-40, Triton X-100, SDS, and CHAPS2 (6). These findings suggest that a broad range of stresses that perturb membrane integrity are responsible for the induction of Drp35 and that Drp35 may be a factor responsible for such general stresses rather than specific anti-biotic stress.

Interestingly, Drp35 possesses calcium-dependent lactonase activity, although it has not been clarified how this activity contributes to the ability of the S. aureus cell to cope with stress (6). In eukaryotic cells, paraoxonase family proteins (PONs) act as lactonases, and these proteins also require calcium ions for their catalytic activity similarly to Drp35 (7-9). Based on these observations, it has been proposed that Drp35 is a bacterial counterpart of eukaryotic PONs (6). PONs are promiscuous enzymes and can hydrolyze not only lactone but also paraoxon, phosphotriester, and esters and thereby inactivate various organophosphates, including insecticides or nerve agents, such as sarin and soman (7, 8). Several studies also proposed a relationship of PONs to diseases, such as antiatherosclerotic activity (8, 10-12). The reaction mechanism of hydrolysis by PONs has been studied by structural analysis, directed evolution, and site-directed mutagenesis (13-15). However, their physiological role has not been determined despite many studies, including molecular characterization, of these proteins. In analogy with the promiscuous function of PONs, Drp35 may play some important role in detoxification of compounds that affect the cytoplasmic membrane, and therefore, elucidating the precise function of Drp35 may provide some insight to overcome staphylococcal pathogenesis. However, Drp35 has not been characterized in detail at the molecular level, including determination of its physiological role. In the present study, we investigated its lactonase activity from a structural view-point. First, we report the crystal structures of Drp35 with and without calcium ions. In the presence of Ca2+, Drp35 has a six-bladed beta-propeller structure with a calcium ion at its center. Comparison of lactonase activity with mutant proteins indicated the significance of residues coordinating to the calcium ion. Structural analysis of the D138N mutant and functional analyses using several mutants suggested that a water molecule coordinated to the calcium ion in the wild type may be essential for catalysis. Based on these findings, we discuss the mechanism of lactonase activity of Drp35.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All enzymes used in genetic engineering were obtained from Takara Shuzo (Kyoto, Japan) and Toyobo (Osaka, Japan). Isopropyl beta-D-thiogalactopyranoside was obtained from Wako Fine Chemicals Inc. (Osaka, Japan). All other reagents were of biochemical research grade.

Construction of Expression Vector for Drp35 and Mutant Proteins—The gene encoding Drp35 was amplified using KOD-Plus DNA polymerase (Toyobo), with S. aureus Mu50 genomic DNA as a template. The NcoI recognition sequence, CCATGG, in the drp35 gene was replaced with CCGTGG, which does not change the amino acid sequence, by the two-stage PCR method using the following primers: Drp35-S (5'-NNNNCCATGGCCATGTCACAACAAGATTTACCTACATTATTTTATAGC-3'), Drp35-AS (5'-CCGCTCGAGTTGAAACTGAAAACTTTGATGACCTTTTGC-3'), Drp35-mut-S (5'-ACAGCTGAACCGTGGCTTGAAATT-3'), and Drp35-mut-AS (5'-AATTTCAAGCCACGGTTCAGCTGT-3') (restriction sites for digestion and ligation are underlined, and mutated nucleotides are indicated in italic type). The PCR products were inserted into the NcoI and BamHI sites of the pET28b vector (EMD Biosciences, San Diego, CA).

All expression vectors for mutant proteins were prepared with a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using synthesized primers and the Drp35 expression vector described above as the template. The correctness of the DNA sequences was confirmed using an ABI 310 Genetic Analyzer (Applied Biosystems, Tokyo, Japan).

Expression and Purification of Drp35 and Mutants—Transformed Escherichia coli strain B834 (DE3), harboring Drp35 expression vector and pT-RIL (Stratagene, Madison, WI), was grown at 37 °C in LB medium supplemented with 50 µgml-1 kanamycin and 34 µgml-1 chloramphenicol until the early stationary phase. To induce expression of the desired protein, isopropyl beta-D-thiogalactopyranoside was added to a final concentration of 0.5 mM, and the culture was continued for 18 h at 25 °C. The selenomethionine derivative of Drp35 was obtained by the same method as described above except using M9 medium supplemented with 1 mM selenomethionine instead of LB medium.

Cells were harvested by centrifugation at 5000 x g for 10 min at 4 °C and then disrupted using a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan) in 50 mM Tris-HCl (pH 8.0), 300 mM NaCl. The cell debris was removed by centrifugation at 40,000 x g for 30 min at 4 °C, and the supernatant was loaded onto a HisTrap column (GE Healthcare Biosciences AB, Uppsala, Sweden) preequilibrated with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl. The column was washed with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and then the adsorbed protein was eluted with 50 ml of a 0-0.5 M gradient of imidazole in 50 mM Tris-HCl (pH 8.0), 300 mM NaCl. Fractions containing Drp35 were dialyzed against 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM EDTA and then further purified on a HiLoad 26/60 Superdex 200-pg column (GE Healthcare Biosciences AB) equilibrated with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM EDTA. Fractions containing the desired protein were collected and used for further experiments.

The Ca2+-bound form of Drp35 and Drp35-D138N mutant were prepared by the same methods as described above except using the following buffers: for cell disruption and equilibration for HisTrap column, 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM CaCl2; for elution from HisTrap column, 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM CaCl2, 500 mM imidazole; for size exclusion chromatography, 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM CaCl2.

For the enzyme assay, Drp35 and mutants were purified by the same method as described above, except for the use of a Hiprep 26/10 desalting column (GE Healthcare Biosciences AB) preequilibrated with 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM CaCl2 instead of dialysis and size exclusion chromatography.

Crystallization of Drp35, Drp35-Ca2+ Complex, and Drp35-D138N Mutant—Purified Drp35 with or without CaCl2 was dialyzed against 20 mM Tris-HCl (pH 7.5) and 20 mM Tris (pH 8.0), with or without 5 mM CaCl2, respectively, and then concentrated up to 20 mg ml-1. Initial crystallization conditions were screened by the sparse matrix method at 20 °C, using a Crystal Screen kit, Crystal Screen 2 kit (Hampton Research, Laguna Hills, CA), Wizard I, and Wizard II (Emerald Biostructures, Bainbridge Island, WA). Crystals of the apo form of Drp35 most suitable for further analyses were grown by the hanging drop vapor diffusion method from 100 mM Tris buffer, pH 8.8, 23% polyethylene glycol 4000, 0.1 M lithium sulfate. Crystals of Drp35 complexed with Ca2+ were grown from 100 mM HEPES (pH 7.6), 1050 mM succinic acid (pH 7.0), 1% polyethylene glycol monomethyl ether 2000. Crystals of the Drp35-D138N mutant were grown from a buffer containing 100 mM HEPES (pH 7.2), 1000 mM succinic acid (pH 7.0), 1% polyethylene glycol monomethyl ether 2000.

X-ray Diffraction—X-ray diffraction of selenomethionine-substituted Drp35 was performed on beamline NW12 at Photon Factory (Tsukuba, Japan) under cryogenic conditions (100 K). For single-wavelength anomalous diffraction (SAD) phasing, a wavelength of 0.97908 Å was chosen on the basis of the fluorescence spectrum of the selenium K absorption edge. The diffraction data of the Drp35-Ca2+ complex and Drp35-D138N mutant were collected on beamline BL44B2 at SPring-8 (Harima, Japan) and on beamline NW12 at Photon Factory (Tsukuba, Japan), respectively. All of the diffraction data were indexed, integrated, scaled, and merged using the HKL2000 program package (16). The data collection and processing statistics are summarized in Table 1.


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  		TABLE 1
X-ray data collection and refinement statistics

 
Structure Solution and Refinement—The initial phasing was achieved by the SAD method with the program SHELX (17). The subsequent phase improvement was performed with the programs SOLVE/RESOLVE (18-22) and DM (23-25). SHELX identified the positions of all 60 selenium sites. The program SOLVE/RESOLVE was used for heavy atom refinement and initial phasing. The initial electron density map was obtained after phase improvement by the program DM with the operator of noncrystallographic symmetry obtained by RESOLVE. A total of 2756 of 3996 residues were built automatically by RESOLVE, and 3792 residues were rebuilt manually using the program LSQKAB (26) as part of the CCP4 suite (27) and the graphics program O (28), based on the initial electron density map. The additional model building, positional energy minimization, and individual B factor refinements were carried out automatically with the program LAFIRE (29). To monitor the refinement, a random 10% subset from all reflections was set aside for calculation of the free R factor (Rfree). After automatic refinement and model fitting by LAFIRE, several cycles of refinement with the program CNS (30) and manual model fitting were carried out, and then the water molecules were located automatically. Due to the abundance of residues in an asymmetric unit against observed reflections, the difference between R and Rfree factor remarkably increased without the noncrystallographic symmetry restriction even in the final step of refinement. Therefore noncrystallographic symmetry restriction was applied to only the regions in which atoms could be well imposed throughout the refinement. The average of r.m.s. deviation of 12 molecules is 0.144 Å. Finally, 3849 residues and 1295 water molecules could be placed in the structure of Se-Drp35 with crystallographic R values and Rfree values of 20.0 and 22.2%, respectively.

The structure of Drp35-Ca2+ complex and D138N mutant were determined by the molecular replacement method using the program MOLREP (31) using the structure of Se-Drp35 as a search probe. To monitor the refinement, a random 10% subset from all reflections was set aside for calculation of the Rfree factor. The positional and individual B factor refinements were carried out automatically with the program LAFIRE. After automatic refinement and model fitting by LAFIRE, several cycles of refinement with the program CNS and manual model fitting were carried out. Finally, the water molecules were picked automatically by the program LAFIRE, and then calcium ions and ligand molecules were placed manually. The crystallographic R values and Rfree values for the Drp35-Ca2+ complex and Drp35-D138N mutant converged to 16.9% (19.7%) and 16.8% (20.2%), respectively.

The stereochemical qualities of the final refined models were analyzed using the program PROCHECK (32). The refinement statistics are summarized in Table 1. Although crystals of Drp35-Ca2+ complex and Drp35-D138N mutant belong to space group P21 in analogy with Se-Drp35, none of the relationship was observed in cell parameters. The packing of noncrystallographically related molecules also does not have a simple correlation.

Enzyme Assay—Lactonase activity was determined as described previously using dihydrocoumarin as the substrate (6). The kinetic parameters for Drp35 were determined in the range of 4.1-9.0 from Lineweaver-Burk plots. Buffers used were acetate (pH 4.1-5.5), MES (pH 5.6-6.8), and Tris-HCl (pH 7.4-9.0). kcat/Km values for each pH value ((kcat/Km)H) were fitted to a bell-shaped model using the equation, (kcat/Km)H = (kcat/Km)max/((10-pH/10-pKa1) + (10-pKa2/10-pH) + 1), where (kcat/Km)max is the pH-independent value, and pKa1 and pKa2 are the apparent pKa values for the acidic and basic groups, respectively.

Differential Scanning Calorimetry (DSC)—All DSC measurements were carried out with a VP-CAPILLARY DSC SYSTEM (MicroCal, Northampton, MA). Proteins were dialyzed against 50 mM acetate, pH 5.6, and 1 mM EDTA or against 50 mM acetate, pH 5.6, and 1 mM CaCl2. The dialysis buffer was used as a reference solution for the DSC scan. Protein samples of 0.60-0.94 mg ml-1 were heated from 10 to 85 °C at a scanning rate of 1 K min-1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Crystal Structure of Se-Drp35 and Drp35-Ca2+ Complex—The crystal structure of selenomethionine-substituted Drp35 was determined at a resolution of 2.4 Å by the SAD method (Table 1). This structural information enabled a molecular replacement method to determine the structure of the Drp35-Ca2+ complex at a resolution of 1.72 Å. The subunit structures of the Drp35-Ca2+ complex are well superposed with those of Se-Drp35 with an averaged r.m.s. deviation of 0.30 Å.

Each monomer consists of five helices ({alpha}1-{alpha}5) and 25 beta-strands (beta1-beta25). These beta-strands form six beta-sheets, five of which are composed of four beta-strands and one of which is composed of five beta-strands (Fig. 1A). These beta-sheets are located in a circular arrangement, resulting in a six-bladed beta-propeller structure. As is often the case with many kinds of beta-propeller protein, a "molecular clasp" tethering the N- and C-terminal regions within a beta-sheet was also observed in two beta-sheets (blades 5 and 6) in Drp35 (33). Interestingly, in all molecules of Se-Drp35, Drp35-Ca2+ complex, and Drp35-D138N mutant (see below), cis-peptide conformation was observed in His232-Glu233, located in the connecting loop between blades 1 and 6 (Fig. 1A).

A structural alignment of Drp35 against all proteins in the Protein Data Bank by secondary structure matching (SSM) showed that the structure of Drp35 was similar to that of diisopropylfluorophosphatase (DFPase) from Loligo vulgaris and serum paraoxonase 1 (PON1) (r.m.s. deviation of 1.87 Å for 264 C{alpha} atoms and 2.50 Å for 237 C{alpha} atoms, respectively), although there was not significant amino acid sequence similarity (BLAST E-scores 4.3 and 1.6 for DFPase and PON1, respectively) (13, 34). These two eukaryotic proteins are classified as phosphotriesterases (EC 3.1.8) and possess a common biological activity (i.e. calcium-dependent hydrolysis activity) (9, 35). Similarly, Drp35 can hydrolyze lactones in a calcium-dependent manner and is a functional counterpart of PON (i.e. Drp35 is related structurally and functionally to DFPase and PON). The cis-peptide conformation found in Drp35 is not observed in the corresponding region in DFPase and PON1. Another apparent structural difference of Drp35 from PON is the absence of a canopy composed of {alpha}-helices that is required for binding of PON to the lipid layer of high density lipoprotein (13). This is consistent with the observation that Drp35 is a soluble cytosolic protein and is not detected in the membrane fraction (6).

The reaction mechanisms for DFPase and PON have been reported. In both enzymes, a pocket at the center of the beta-propeller acts as a substrate entrance tunnel in which substrates are captured and reacted. Drp35 also has a pocket at the identical position (Fig. 1C), suggesting that it would hold the active site (see below).

In the structure of Drp35-Ca2+ complex, two calcium ions (Ca1 and Ca2) were bound to one molecule of Drp35 (Fig. 1A). The averaged distance between Ca1 and Ca2 is 17.0 Å. Ca1 was bound at the bottom of the pocket located at the center of the beta-propeller, where eight oxygen atoms derived from the side chains of Glu48, Asp138, Asn185, Asp236, Ser237, and three water molecules coordinated to Ca1 (Fig. 1, B and C). One of the water molecules was bound not only to Ca1 but also to O{delta} in Asp138. The average distances between Ca1 and each ligated oxygen atom were as follows: 2.48 Å for O{epsilon} in Glu48, 2.52 Å for O{delta} in Asp138, 2.37 Å for O{delta} in Asn185, 2.36 Å for O{delta} in Asp236, 2.41 Å for O{gamma} in Ser237, and 2.56, 2.42, and 2.38 Å for three water molecules. The other calcium, Ca2, is bound on the surface, in which the carbonyl oxygens from Thr133, Ser110, Asp130, Tyr135, and Gly112 and O{gamma} from Thr133 were bound to Ca2. The average distances between Ca2 and each ligated oxygen atom were as follows: 2.53 Å for oxygen in Thr133, 2.30 Å for oxygen in Ser110, 2.52 Å for oxygen in the main chain of Asp130, 2.50 Å for oxygen in Tyr135, 2.47 Å for oxygen in Gly112, and 2.51 Å for O{gamma} in Thr133. Although all coordinating atoms were oxygens as in Ca1, none of the carboxylate groups, which are the most frequently observed groups as ligands in Ca2+-protein complexes (36), participated in the Ca2 binding.

In crystal structures of DFPase and PON1 reported previously, a catalytically important calcium ion was observed at the identical positions to Ca1. The structural comparison of the Ca1 binding site in Drp35 with those in DFPase and PON1 shows that they have similar coordination geometries (Fig. 2), where three of five residues bound to Ca1 (i.e. Glu48, Asn185, and Asp236) were conserved among the three proteins, Drp35 (Fig. 2A), DFPase (Fig. 2B), and PON1 (Fig. 2C). Glu48, Asn185, and Asp236 in Drp35 correspond to Glu53, Asn224, and Asp269 in PON1 and Glu21, Asn175, and Asp229 in DFPase, respectively. However, two other residues, Asp138 and Ser237, were not conserved. Ser237 in Drp35 is replaced by Asn270 in PON1, and none of the residues coordinated here in DFPase. Asp138 in Drp35 is replaced with Asn in both PON1 and DFPase. DFPase and PON1 possess a structural calcium ion in addition to the catalytic calcium at an identical position, near the center of the beta-propeller (13, 34). The distances between these two calcium ions are 7.4 and 9.4 Å, respectively. However, no calcium ion was observed at the corresponding site in Drp35.

Structural and mutational analyses suggested that His287 and Glu37 around the calcium binding site are catalytic residues in DFPase (34, 37). In PON1, two histidine residues, His115 and His134, have been suggested to be catalytic residues, acting as a general base and proton donor, respectively (13, 15). Fig. 2 shows the catalytic residues in DFPase (Fig. 2B) and PON1 (Fig. 2C) and residues at corresponding positions in Drp35 (Fig. 2A). Interestingly, none of the catalytic residues in DFPase and PON1 are conserved in Drp35; His287 and Glu37 in DFPase are replaced with Asn299 and Phe64, and His115 and His287 in PON1 are replaced with Ala90 and Leu105. These results strongly suggest that Drp35 has a catalytic mechanism distinct from two other functionally and structurally related proteins.


Figure 1
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  		FIGURE 1.
Crystal structure of Drp35-Ca2+ complex. A, ribbon diagram of the Drp35-Ca2+ complex. The ribbon model is colored according to the sequence from blue at the N terminus to red at the C terminus. Two calcium ions binding to Drp35 (Ca1 and Ca2) are shown as pink balls. The cis-conformation residues, His232-Glu233, are shown as sticks. B, close-up view of the Ca1 binding site. Ca1 is shown as a pink ball, and residues coordinating to Ca1 are shown as sticks. Water molecules binding to Ca1 are also shown as blue balls. C, a surface view of the pocket in which Ca1 is bound at the bottom. Red and blue represent negative and positive charges, respectively. Two calcium ions are shown as green balls. Phe64 is also shown as orange balls.

 
Effects of Mutations into Conserved Residues on Lactonase Activity—To acquire insight into the active site of Drp35, we analyzed the structures of crystals grown or soaked in buffer containing the competitive inhibitor compounds, coumarin, benzamidine, and 8-quinolinol. However, we could not detect the clear electron density derived from the inhibitor compounds in any case despite the obvious electron density of Ca2+. Then we focused on residues conserved among proteins homologous to Drp35 in other bacteria. A sequence alignment among seven homologous proteins proposed 27 residues as completely conserved residues (Fig. 3). Alanine-substituted mutant proteins of these residues were prepared, and their lactonase activities were compared. There were problems in preparation of three mutants, Gly231, Asp241, and Val248. In the case of Gly252, the G252V mutant was used, because we could not construct an expression vector for G252A for unknown reasons. Fig. 4A shows the activity of each mutant protein relative to wild-type Drp35. Some fluctuations were found in the activities of several mutant proteins, as is also the case with PON (14). Marked decreases were observed in six mutants: E48A, D139A, D152A, N185A, G186A, and D236A. As described above, Glu48, Asn185, and Asp236 directly coordinated to Ca1 in the structure of the Drp35-Ca2+ complex. In addition, Asp139 and Gly186 are neighboring residues to Ca2+-binding residues, Asp138 and Asp185. The distances between C{alpha}s of Asp139 and Gly186 and Ca1 are 5.94 and 5.28 Å, respectively. Although Asp152 is neither a Ca1-binding residue nor a neighboring residue, it is located rather close to Ca1; the distance between the C{alpha} atom of Asp152 and Ca1 is 10.7 Å. These results suggest the importance of Ca1 for catalytic activity of Drp35, as reported for calcium ion binding at the identical position in DFPase and PON1. This was strongly supported by the pocket structure of Drp35, which is generally observed for substrate binding sites in enzymes (38). Ca1 binding at the bottom of the pocket also suggests a connection between the possible active site and the substrate entrance tunnel (Fig. 1C).

Effects of Mutations into Residues around the Calcium Binding Site—We investigated residues located around the Ca1 binding site by alanine scanning (Fig. 4B). Mutated residues could be classified into three groups: residues participating directly in binding to Ca1 (gray bars in Fig. 4B), those comprising the tunnel described above (closed bars in Fig. 4B), and those located in the interior of the protein, where structural calcium was bound in DFPase and PON1 (open bars in Fig. 4B). A comparison of lactonase activity showed that significant inactivation occurred by replacing residues that bind directly to Ca1. The extent of the decrease in the Ser237 to Ala mutant was weaker, although for the other four Ala mutants the decrease was striking. Mutations in residues categorized into two other groups did not bring about noticeable decreases in activity except for Phe153, in which the activity was 7.6% of wild type. It should be noted that lactonase activity increased up to 2.5-fold that of the wild-type enzyme when Phe64, which is the structural counterpart of the catalytic residue Glu37 at the entrance of the tunnel of DFPase, was substituted by alanine. In addition to F64A, increases in lactonase activity were observed in R281A, E302A, K93A, and C238A.


Figure 2
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  		FIGURE 2.
Comparison of the Ca1 binding site in DFPase and PON1. A, Drp35 from S. aureus. B, DFPase from L. vulgaris (Protein Data Bank code 1E1A). C, serum paraoxonase 1 (Protein Data Bank code 1V04). The catalytic calcium ions and their coordinating residues are represented as green balls and sticks, respectively. The residues whose carbon atoms are colored green are conserved among three proteins. The catalytic residues in DFPase (B) and PON1 (C) and the residues located in identical positions to the catalytic residues of DFPase and PON1 in Drp35 (A) are shown as pink sticks. Asp152 and Phe153 are also shown.

 
Effects of Substituting a Variety of Amino Acid Residues for Calcium-binding Residues—The results described above suggested that residues participating in calcium binding except Ser237 strongly influence the lactonase activity. To test whether the integrity of the coordinated network with the calcium ion is critical for lactonase activity, these residues were substituted to other residues as follows: Glu was substituted to Ala, Ser, and Gln; Asp was substituted to Ala, Ser, and Asn; and Asn was substituted to Ala, Ser, and Asp. Fig. 4C shows the resultant activities of these mutants relative to the wild-type enzyme. The replacement of Glu48, Asp138, and Asp236 with other amino acids led to complete abolition of the activity. In the case of Asn185, which is a completely conserved residue in homologous proteins, mutations to Ala or Ser resulted in inactivation, but N185D maintained a certain degree of activity as the S237A mutant. Based on these results, the Ca1-binding residues could be divided into two groups according to their influence on the enzymatic activity; one group includes residues with a strong influence, Glu48, Asp138, and Asp236, whereas the other includes Asn185 and Ser237, the efficacy of which was not so strong.

Evaluation of the Calcium Binding of Drp35, E48Q, D138N, and D236N Mutants by Differential Scanning Calorimetry—To evaluate whether the substitutions in Glu48, Asp138, and Asp236 abolish binding to the calcium ion, the thermostabilities of wild-type Drp35, E48Q, D138N, and D236N were measured by differential scanning calorimetric assay (Fig. 5). Under control conditions without calcium ions, each protein exhibited similar thermograms with a peak between 55.8 and 58.2 °C (Fig. 5A). However, clear differences were observed in the presence of 1 mM CaCl2 (Fig. 5B). The thermogram of the wild type exhibited a peak with the highest value of 70.4 °C, followed by D138N with a peak of 66.6 °C, D236N with a peak of 60.8 °C, and E48Q with a peak of 58.3 °C. The increases in Tm value upon the addition of calcium ions were 12.2 °C for wild type, 9.6 °C for D138N, 5.0 °C for D236N, and 0.3 °C for E48Q. Since the increase in Tm is thought to be due to the structural stabilization caused by calcium ion binding, these results suggest that 1) the marked decreases in enzymatic activity seen in E48Q and D236N are due to loss of the calcium ion and that 2) D138N can bind the calcium ion similarly to the wild type despite the loss of activity. Taken together with the observation that Asp138 is the calcium-binding residue that is not structurally conserved among Drp35, PON, and DFPase (Fig. 2), we considered that Asp138 may be involved in the characteristic catalytic mechanism of Drp35.


Figure 3
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  		FIGURE 3.
Sequence alignment of Drp35 with its homologues. Completely conserved residues are highlighted in red, and conservatively mutated sites are shown in light blue. S. epidermidis, Staphylococcus epidermidis; B. japonicum, Blepharisma japonicum; B. bronchiseptica, Bordetella bronchiseptica; X. fastidiosa, Xylella fastidiosa; Z. mobilis, Zymomonas mobilis.

 
Structural Analysis of D138N Mutant—We solved the crystal structure of the D138N mutant. The crystallographic parameters are not changed from those of the Drp35-Ca2+ complex, and the asymmetric unit contained six molecules of D138N mutant protein (Table 1). Crystals were grown in buffer containing 5 mM calcium ions, and the catalytic calcium was clearly observed as in the wild type with sufficient occupancy in all six molecules, consistent with the results of differential scanning calorimetric analysis. There were no noticeable conformational changes for residues binding to the calcium ion except for the mutated residue, Asn138 (Fig. 6). The carbonyl group in the side chain of Asn138 was rotated by about 15° compared with Asp138 in the wild type. In addition, the water molecule bound to both O{delta} in Asp138, and the calcium ion in the wild-type enzyme (Fig. 6, Water1) disappeared in three of six D138N molecules in the asymmetric unit, although two other water molecules that were also bound to the calcium ion as in the wild type (Fig. 6, Water2 and Water3) were observed. Even in the three molecules of D138N mutant in which water1 could be observed, the electron density corresponding to water1 was not as clear in comparison with the other two water molecules or that observed in the wild-type structure. It is obvious that the occupancy of water1 was noticeably reduced by replacing Asp138 with Asn. The distance between water1 and N{delta} in Asn138 (average value among three molecules) was 2.84 Å, although that of O{delta} in Asp138 in wild type was 2.61 Å.

pH Dependence of Enzyme Catalysis—Acidic pKa values of 7.4 and 6.8 have been reported for PON1 and DFPase, both of which use histidine as the catalytic residue (15, 37). On the other hand, our mutational and structural analyses in Drp35 suggested that the catalytic residues would be acidic residues rather than histidine. If this is true, the pKa of Drp35 would be lower than those of PON1 and DFPase. We measured the pKa value of Drp35 to examine this prediction. Fig. 7 shows the resultant kcat/Km versus pH plot exhibiting a bell-shaped curve with the peak near pH 6.0, which could be fitted to a bell-shaped model with the equation described under "Experimental Procedures" with acidic and basic pKa values of 5.4 and 6.7, corresponding to pKa1 and pKa2 in the equation, respectively. The acidic pKa of 5.4 is, as expected, 2.0 and 1.4 lower than those reported previously for PON1 and DFPase, respectively. In the high pH environment, Drp35 exhibited a decrease in specific activity with an acidic pKa value of 6.7 for as yet undetermined reasons, which was also observed for PON1, where the pKa value was 9.8.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Active Site and Deduced Catalytic Residue—The three-dimensional structure of Drp35, a six-bladed beta-propeller structure, is similar to those of DFPase and PON1, which are functionally related proteins despite the lack of significant amino acid sequence similarity (13, 34). These proteins have the common enzymatic feature of calcium dependence (9, 35). Two calcium ions were observed in the Drp35-Ca2+ complex, one of which (Ca1 in Fig. 1) was present in the identical position in two other proteins (i.e. the center of the beta-propeller). From the results of the present study, we concluded that the Ca1 binding site constitutes an active site, as is the case in both DFPase and PON1.

As described above, we could not reveal any structure complexed with inhibitors. However, in these structures, the Fo - Fc map showed a certain electron density that could not be assigned to any compound at the bottom of the tunnel. Interestingly, this electron density was surrounded by Asp138, Asp236, and the calcium ion (data not shown). In analogy with the other enzymes whose active site can be easily complexed with certain compounds (13, 39-41), this observation also supports our conclusion that the active site is positioned around the calcium ion.

The structural comparison focusing on the residues binding to the catalytic calcium among Drp35, DFPase, and PON1 revealed that coordination of the calcium ion was also quite similar, and three of five residues binding to the catalytic calcium ion, Glu48, Asn185, and Asp236, are conserved among the three proteins (Fig. 2). Despite these structural similarities, the catalytic residues identified in DFPase and PON1 do not exist in Drp35 (Fig. 2), suggesting that the catalytic mechanism of Drp35 is distinct from these enzymes. This was strongly supported by the enhancement, rather than reduction, of the enzymatic activity due to the substitution of Phe64, which is the structural counterpart of the catalytic residue in DFPase (Figs. 2 and 4B).


Figure 4
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  		FIGURE 4.
Changes in lactonase activity by mutation. The activity of each mutant protein relative to the wild-type enzyme is shown. A, results of mutation in completely conserved residues. B, results of mutation in residues located around Ca1. Enzymes with mutations of the residue binding directly to Ca1, those composing the tunnel leading toward Ca1, and those located in the interior of the protein are shown as gray, closed, and open bars, respectively. C, the results of mutations in the residues coordinating to Ca1.

 
The five residues coordinating to the catalytic calcium could be divided into two groups; Glu48, Asn185, and Asp236 are structurally conserved in PON and DFPase, whereas Asp138 and Ser237 are not. On the other hand, mutations in Glu48, Asp138, and Asp236 resulted in marked inactivation irrespective of the substitution residues, although the influences of mutations in Ser237 and Ans185 were rather weak. DSC analysis revealed that the marked inactivation in E48Q and D236N was due to loss of the calcium ion. It was also reported for DFPase that E21Q and D229N, which correspond structurally to Glu48 and Asp236 in Drp35, lacked the calcium ion (34). In contrast, the results of DSC and structural analysis of D138N indicated that the decrease in catalytic activity was not due to the loss of the calcium ion and was probably due to the disappearance of one water molecule, designated water1. These results suggest a probable catalytic mechanism involving water1 (see below), and its binding would be finely tuned by Asp138.

Thus, Asp138 is an intriguing residue in that it is not conserved structurally among the three proteins but is strongly correlated with the catalytic activity of Drp35. Considering the absence in Drp35 of the catalytic residues in PON1 and DFPase, Asp138 seems to specify the unique catalytic mechanism of Drp35. This prediction may also be supported by the acidic pKa value of 5.4. Asp139 and Asp152, of which substitution for Ala caused significant inactivation (Fig. 4A), can be other candidates for this pKa value. Although these locate around Ca1, they would not be the catalytic residue, because 1) Asp139 is located behind Ca1, and it would not be able to interact with a substrate directly unless catalytic Ca2+ goes away, and 2) the side chain of Asp152 does not face the bottom of the pocket in which other essential residues and Ca1 are located.


Figure 5
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  		FIGURE 5.
Differential scanning calorimetry of Drp35, E48Q, D138N, and D236N. Shown are heat capacity curves in 50 mM acetate, pH 5.6, and 1 mM EDTA (A) and in 50 mM acetate, pH 5.6, and 1 mM CaCl2 (B). Solid lines, wild type; dotted lines, E48Q; dashed lines, D138N; dashed and dotted lines, D236N.

 


Figure 6
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  		FIGURE 6.
Superposition of the residues binding to Ca1 of wild-type enzyme with the D138N mutant. The residues binding to Ca1 are represented as sticks. The carbon atoms in the wild-type enzyme are colored yellow, and those in D138N mutant are green. The water molecules observed in wild-type enzyme are shown as blue balls, and those in D138N are shown as red balls. Ca1 is shown as a green ball.

 


Figure 7
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  		FIGURE 7.
The effect of pH on kcat/Km of lactonase activity of Drp35. The data were fitted to a bell-shaped model with the equation described under "Experimental Procedures."

 
The mechanism responsible for the basic pKa value of 6.7 is unclear, but it indicates that the solved structure should represent the form inactivated by the basic environment, because crystallization was performed at pH higher than 6.7. The coordination of hydroxyl groups of the water molecules to the catalytic calcium may be affected by the pH value to influence the activity. It should also be noted that the pKa value of 6.7 would not be attributable to imidazole groups, because mutations in any of the histidine residues located around the active site did not cause significant inactivation (Fig. 4).

Proposed Reaction Mechanism of Drp35—Based on the findings described above, we propose a possible reaction mechanism of Drp35 as follows (Fig. 8). First, the water molecule binding to Ca1 and Asp138 is activated by deprotonation. The substrate enters the tunnel and binds to Ca1, and the activated water molecule is then located beside the substrate. The water would attack the carbon atom in the carbonyl group of the substrate and cause hydrolysis. Protonation accompanied by hydrolysis is thought to be mediated by Asp236, as suggested by the steric arrangement around Ca1. This was also supported by the observation that the unassigned electron density beside the Ca1 is surrounded by Asp138, Ca1, and Asp236. Although the pKa value of the carboxyl group in proteins is usually within the range from 2 to 5.5, it was reported that the aspartic acid residue acts as a proton donor with a pKa value of 6.5 and 7.8 in two calcium-dependent hydrolases, nucleoside hydrolase and {alpha}-mannosidase, respectively (42, 43). In analogy with this, it is possible to suppose that Asp236 is protonated within the pH range optimal for the catalytic activity of Drp35, embedded in its unusual environment.

The calcium ion is found in proteins, such as calmodulins and other Ca2+ sensor proteins (44), and it plays a role in structure-forming switching control. It is also involved in stabilizing protein structure (35, 45, 46). In addition, calcium ion plays roles in substrate binding or catalytic reaction in some enzymes, including DFPase (34), PON1 (15), glycosidase (43), nuclease (42, 47), and phospholipase A2 (48). It should be noted that the proposed catalytic mechanism for Drp35 is similar to the established mechanism in nucleoside hydrolase (49), {alpha}-mannosidase (43), and glucoamylase, where a water or carboxyl group coordinating to the calcium ion indeed participates directly in the hydrolytic reaction. Nucleoside hydrolase has been studied extensively by structural and biochemical analyses, and it was reported that the water molecule bound to both the catalytic calcium and O{delta} from Asp10, which also bound to catalytic calcium via the other O{delta} atom, is activated and attacks the substrate (49). Protonation of intermediates by the carboxyl group binding the catalytic calcium via O{delta} was recently identified for glucoamylase.3 In this enzyme, two carboxyl groups binding directly to calcium act as catalytic residues, and the calcium ion directly binds the substrate. One carboxyl group, which acts as a general base, activates the water molecule essential for nucleophilic attack, and the other one, which acts as a general acid, protonates the intermediate. These reports also constitute a rationale for the Asp138-dependent mechanism proposed for Drp35. Although such a reaction mechanism has been reported previously for only a few proteins, it may be a typical mechanism of hydrolysis.


Figure 8
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  		FIGURE 8.
Schematic representation of proposed mechanism for lactonase activity of Drp35. A water molecule bound to Ca1 and Asp138 is activated by Asp138 and Ca1 (left). The generated hydroxyl group attacks the carbon atom in the carbonyl group of the substrate, and the oxygen atom whose covalent bond is broken is protonated by Asp236 (center). 3-(2-Hydroxyphenyl)propionic acid is generated.

 
In the proposed model, the calcium ion plays the critical role in the catalysis. This is consistent with the fact that EDTA inhibits the activity of Drp35 (6). It is known that some enzymes, such as nucleoside hydrolase and {alpha}-mannosidase, specifically require the Ca2+ (50, 51), whereas it can be substituted with other cations without abolishing the catalytic activity in phospholipase A2 (48). Drp35 is also compatible with other divalent cations, such as Mg2+ or Mn2+ (data not shown). Elucidation of the catalytic mechanism(s) involving such distinct kinds of cations will be important future work to know the cation specificity of this catalysis model.

Biological Implications—Drp35 is thought to be related to stress adaptation or clearance, and it apparently possesses lactonase activity (6). However, it is still unclear how this activity contributes to the mechanisms of coping with stresses, and the nature of the genuine substrate in vivo has yet to be determined. The F64A mutant of Drp35 showed 2.5-fold higher activity than the wild-type enzyme. Phe64 is located at the entrance of the tunnel that connects to the active site. This would reflect the disagreement of the substrate used for measuring enzymatic activity (i.e. dihydrocoumarin) with its true substrate in vivo. The activation may be caused by fortuitous optimization of the structure for dihydrocoumarin. Replacement of the bulky phenylalanine residue with alanine may cause increases in plasticity and the dimensions of the entrance. The true substrate may be a smaller or stalkier compound than 3,4-dihydrocoumarin that could easily reach the catalytic center even in the wild-type enzyme. The negatively charged surface of the tunnel also suggests that the substrate would have a positive charge. In the future, extensive compound screening should be conducted to identify the in vivo substrate. This will provide critical insight into the function of Drp35 and facilitate the development of means to overcome hospital-acquired S. aureus infection.

Conclusions—Structural analyses of Drp35 revealed a six-bladed beta-propeller structure with a calcium ion at the center. Mutation analyses indicated the significance of the calcium ion and its coordinating residues. The crystal structure of the D138N mutant showed the disappearance of a water molecule in the active site. Based on these findings, a possible mechanism was proposed for the lactonase activity in which the calcium ion and the coordinating aspartate residues participate.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2DG0, 2DG1, and 2DSO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by grants-in-aid for younger scientists from the Ministry of Education, Science, Sports, and Culture of Japan (to Y. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 81-11-706-3221; Fax: 81-11-706-4905; E-mail: tanaka{at}castor.sci.hokudai.ac.jp.

2 The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PON, paraoxonase family protein; MES, 4-morpholineethanesulfonic acid; DSC, differential scanning calorimetry; DFPase, diisopropylfluorophosphatase; r.m.s., root mean square. Back

3 M. Kitamura, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank M. Takano and R. Kuwahara (Hokkaido University) for technical assistance. We also thank M. Kitamura of Hokkaido University and Dr. K. Suzuki of the University of Tokyo for helpful suggestions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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