Crystal Structure of D-Aminoacylase from Alcaligenes faecalis DA1 A NOVEL SUBSET OF AMIDOHYDROLASES AND INSIGHTS INTO THE ENZYME MECHANISM*

From the ‡Department of Life Science, §Institute of Biochemistry, National Yang-Ming University, Taipei 11221, Taiwan, ¶Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan, Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, **Department of Biochemistry, National Taipei College of Nursing, Taipei 11219, Taiwan, and ‡‡X-ray Structural Biology Group, Synchrotron Radiation Research Center, Hsinchu 30077, Taiwan

N-Acyl-D-amino acid amidohydrolases (D-aminoacylases, EC 3.5.1.14) catalyze the zinc-assisted hydrolysis of N-acyl-Damino acids to produce the corresponding D-amino acids, which are intermediates in the preparation of pesticides, bioactive peptides, and antibiotics. Recently, D-amino acids have been found in bacteria, plants, and animals, and their physiological functions have received increased attention. Production of Lamino acids by optical resolution using L-aminoacylase immo-bilized on DEAE-Sephadex has been used in industry. Therefore, production of D-amino acids using D-aminoacylase has commercial importance.
The high degree of global structure and the metal center similarity of phosphotriestase, adenosine deaminase, and urease have been noted once these structures were solved (13). Subsequent superposition of these three protein structures by Holm and Sander (11) revealed a common ellipsoidal (␤␣) 8barrel with conserved metal ligands, four histidines and one aspartate, at the C-terminal ends of strands ␤1 (HXH), ␤5 (His), ␤6 (His), and ␤8 (Asp), and led to discovery of the ␣/␤barrel amidohydrolase superfamily. The five metal ligands are strictly conserved and define a subtle but sharp sequence signature in this superfamily.
On the basis of the metal centers in the known crystal structures, the superfamily has been divided into two subsets: urease (13), phosphotriesterase (14), phosphotriesterase homology protein (15), dihydroorotase (16), and dihydropyrimidinase (17), containing a binuclear center; and adenosine deaminase (18) and cytosine deaminase (19) with a mononuclear center (see below). We report here that D-aminoacylase can be described as a defining member of a novel subset based on its unusual metal center. The enzyme structure also suggests the substrate specificity and the catalytic mechanism.

EXPERIMENTAL PROCEDURES
The recombinant protein was expressed, isolated, and crystallized as described previously (20). All x-ray data were collected at 100 K. The crystals belong to space group P2 1 2 1 2 1 , with cell dimensions a ϭ 60.2 Å, b ϭ 76.6 Å, and c ϭ 135.3 Å. The structure was solved using the Se-SAD methods (20) and was then refined by using CNS (21). The x-ray data were collected at beamlines BL6A and BL18B at the Photon Factory, Tsukuba, Japan, and BL41XU and BL12B2 at SPring-8, Sayo, Japan. The refinement parameters are presented in Table I. More than 91% of the residues are in the most favored regions, with the remaining ones in the additional allowed regions except His 250 , Thr 290 , and Thr 406 due to hydrogen bond interactions. Figs. 1, 3B, and 4 were generated by MOLSCRIPT (22)

RESULTS AND DISCUSSION
The Overall Structure-The DA1 D-aminoacylase has 483 amino acids, and the current model contains residues 7-480 with clear electron density. The protein is composed of a small ␤-barrel (residues 7-61 and 425-480) and a catalytic ␣/␤barrel (residues 62-414) (Fig. 1). There are two insertions in the ␣/␤-barrel: residues 147-165 between the ␤3 strand and ␣3-helix, forming a helix and a random coil, and residues 285-347 between ␤7 and ␣7, forming two helices and four ␤ strands. The latter 63-residue insertion across the active site is involved in the substrate-mediated conformational change (see below). There are two large loops between ␤2 and ␣2 (residues 99 -127) and between ␤8 and ␤8 (residues 366 -398).
Structural similarity search by DALI (25) revealed that the closest structural matches of D-aminoacylase are seven members of the recently identified ␣/␤-barrel amidohydrolase superfamily mentioned above (13)(14)(15)(16)(17)(18)(19), in particular, urease and cytosine deaminase, with r.m.s. 1 deviations of 2.9 Å (325 C␣ atoms with 18% sequence identity) and 3.8 Å (330 C␣ atoms with 12% sequence identity), respectively. Structural comparison reveals that the ␤-strands in both the small ␤ barrel and the catalytic ␣/␤-barrel correspond closely, whereas the external helices and surface-exposed loops diverge significantly. Despite the apparent lack of sequence similarity, the eight ␤-strands (56 structurally equivalent residues) of the ␣/␤-barrels overlay within 1.2-2.0 Å r.m.s. deviations ( Fig. 2A). On the other hand, the size of the small ␤-domain, comprising both the N and the C termini, varies greatly in different structures. The ␤-strands of the ␤-domains (48 structurally equivalent residues) overlay within 1.0 -1.3 Å r.m.s. deviations (Fig. 2B). The small ␤-barrel does not contribute any residues to the active site and appears to play a structural role.
A Mononuclear Metalloenzyme with a Binuclear Active Site-Unexpectedly, the active site contains only one tightly bound metal ion (Fig. 3A). On the basis of the atomic absorption analysis (10) and the zinc anomalous data, this metal ion is assigned as zinc. The zinc ion is tetrahedrally coordinated by Cys 96 S ␥ (2.24 Å), His 220 N ␦1 (2.08 Å), His 250 N ⑀2 (2.04 Å), and an acetate molecule, ACT1 O 2 (2.04 Å), from the crystallization solution.
There is another potential metal-binding site, surrounded by His 67 , His 69 , Cys 96 , Asp 366 , and ACT1, and similar to the metalbinding sites in the ␣/␤-barrel amidohydrolase superfamily (13)(14)(15)(16)(17)(18)(19). A small electron density at this site is observed after structural refinement (Fig. 3A). The electron density at this site becomes much stronger in the crystals soaking with 100 mM zinc acetate. In addition, the zinc content of the purified recombinant DA1 D-aminoacylase was measured to be between 1.3-1.5 g⅐atom per mole of enzyme (10). Therefore, the weak electron density is assigned as zinc, which is tetrahedrally ligated by His 67 N e2 (2.01 Å), His 69 N ⑀2 (2.05 Å), Cys 96 S ␥ (2.19 Å), and ACT1 O 1 (2.26 Å), and separated from the tightly bound zinc by 3.1 Å. The crystallographic refinement resulted in an occupancy of 0.25 with B-factor of 19.3 Å 2 for the loosely bound zinc ion and an occupancy of 1.0 with B-factor of 7.3 Å 2 for the tightly bound zinc.
Thus, the DA1 D-aminoacylase binds two zinc ions with widely different affinities. Only the tightly bound zinc is required for the enzyme activity, because the isolated enzyme exhibits significant activity. Addition of extrinsic zinc ions does not enhance the enzyme activity. A large excess of zinc ions even strongly inhibits the enzyme activity (data not shown). Therefore, the DA1 D-aminoacylase is a mononuclear enzyme but contains a binuclear active site, bearing an interesting analogy to the ␤-lactamase from Bacillus cereus (26 -28).
Even though ␤-lactamases share significant sequence identity (34%) with highly conserved metal ligands, the enzyme from Bacteroides fragilis has a binuclear zinc center with similar metal affinities (K d ϳ10 M), whereas the B. cereus enzyme binds zinc ions with very distinct affinities (K d ϳ1 M and 25 mM, respectively). The crystal structures suggested that the weak metal binding may be due to the local electrostatic environment (26), and the B. cereus enzyme functionally behaves as a monozinc enzyme and may be an evolutionary intermediate between the mono-and bi-zinc metallo-␤-lactamases (27,28).
A Novel Subset of the ␣/␤-Barrel Amidohydrolase Superfamily-To date, there are two subsets in the ␣/␤-barrel amidohydrolase superfamily based on the metal centers with four conserved histidines and one aspartate (Fig. 3B). In the binuclear subset (13)(14)(15)(16)(17), the more buried metal ion (␣ site) is coordinated by the first two conserved histidines from the common zincbinding HXH motif (29), the conserved aspartate, and two bridging ligands, whereas the more solvent-exposed metal ion (␤ site) is chelated by the other two conserved histidines and the bridging ligands, consisting of a carboxylated lysine (or a glutamate) and one water molecule (or a hydroxide ion). On the other hand, in the second subset (18,19), the metal is bound only at the ␣ site ligated by the first three conserved histidines and one water molecule.
In D-aminoacylase, one zinc ion binds strongly at the ␤ site, and the other binds weakly at the ␣ site. This is the first example of a cysteine residue (Cys 96 ) that coordinates to a zinc ion in this superfamily (Fig. 3B). Mutational and atomic absorption spectroscopic studies revealed that this cysteine residue contributes the most toward the interactions with the zinc ions among the ligands, because the mutant C96A shows the least zinc binding affinity (10). Therefore, the unique metal center of D-aminoacylase defines a novel subset, in which two metal ions bind to the binuclear metal center with different affinities and are bridged by a thiolate ligand (cysteine) instead of a carboxylate ligand (carboxylated lysine or glutamate).
Structural superposition demonstrates that the metal centers in the same subset are virtually identical (10, 17, 18). Remarkably, superposition of the metal centers in different subsets, i.e. D-aminoacylse, urease, and cytosine deaminase, reveals that the metal ligands are also at the similar spatial positions, with Cys 96 occupying the position of the carboxylated lysine (Fig. 3B). In cytosine deaminase and adenosine deaminase, the third conserved histidine compensates the missing carboxy-lated lysine. Approximately two-thirds phosphotriesterase homology proteins such as those from human, mouse, rat, fly, Bacillus, Salmonella, and Escherichia coli, use a glutamate instead of the carboxylated lysine, resulting a larger structural difference at the ␤4 strand in the ␣/␤-barrel (15; Fig. 2A). The zinc-zinc distance of 3.1 Å in D-aminoacylase is similar to the nickel-nickel  3. The metal center. A, the 2F o Ϫ F c electron density map in the zinc center contoured at the 3 level and is shown in green, and the weak density in the 2F o Ϫ F c map for the loosely bound zinc ion contoured at the 2 level and is shown in purple. The structural refinement revealed that the enzyme binds two zinc ions with very different affinities. B, superposition of the bi-nickel center in urease (Protein Data Bank code 1UBP), the mononuclear iron center in cytosine deaminase (Protein Data Bank code 1K6W), and the bi-zinc center in D-aminoacylase, shown in red, blue, and green, respectively. The ␣ metal-binding site is more buried, while the ␤ site is more solvent-exposed. The residue numbering is labeled in the same color for each protein. The critical hallmark for the binuclear subset is a carboxylated lysine residue serving as a bridging ligand. A cysteine residue (Cys 96 ) in D-aminoacylse, and the third conserved histidine (His 214 ) in cytosine deaminase, compensate the missing carboxylated lysine. FIG. 1. The A. faecalis D-aminoacylase structure. A, ribbon diagram of the C␣ backbone. The structural fold contains a small ␤-barrel comprising both the N and the C terminus (residues 7-61 and 425-480), a central ␣/␤-barrel (residues 62-414), with a 63-residue insertion (residues 285-347). The small ␤-barrel may be responsible for the structural stabilization, the ␣/␤-barrel for catalysis, and the insertion for substrate-mediated conformational switch. The tightly bound zinc ion, the metal ligands, and two acetate molecules are shown as a pink sphere and ball-and-stick representation. B, a close-up view from the top of the elliptically distorted ␣/␤-barrel. The tightly bound zinc ion is chelated by Cys 96 , His 220 , His 250 , and the first acetate. distance of 3.1 Å in the ␤-mercaptoethanol (␤-ME)-inhibited Bacillus pasteurii urease (30), but it is significantly shorter than those (3.4 -3.8 Å) in the other crystal structures of the binuclear members (13)(14)(15)(16)(17)31). A screening of the Cambridge Structural Data base reveals that the bridging thiolate sulfur atoms would shorten the di-metal distance. Therefore, the shorter di-metal distance in D-aminoacylase and in ␤-ME urease can be considered as an intrinsic property of the metallic core.
The Putative Substrate-binding Site-Two acetate molecules from the crystallization solution are observed in the active site region (Fig. 4A) On the basis of three assumptions, the preferred substrate N-acetyl-D-methionine was modeled into the active site (Fig.  4B). First, the carboxylate group occupies the position of that of ACT2 because of the extensive interactions mentioned above. Second, the side chain binds at the hydrophobic pocket. And third, according to the structural studies of other ␣/␤-barrel amidohydrolase superfamily members (13)(14)(15)(16)(17)(18)(19)31), the amide oxygen atom coordinates the ␤ ion (which is likely at the ACT1 O 2 position), and the amide carbon atom must be in close proximity to the active water molecule (the ACT1 O 1 position). After modeling, the energy minimization was performed by CNS (21) as the structural refinement.
The model of the bound substrate reveals that the carboxylate and the amide oxygen atoms occupy the positions of acetate oxygen atoms as expected. The amide nitrogen forms a hydrogen bond with Ser 289 O (2.8 Å), and the amide carbon is in close proximity to the predicted water molecule. The side chain packs into the hydrophobic pocket surrounded by Thr 290 , Phe 191 , Lys 252 , Met 254 , and Met 347 , in which Leu 298 , Tyr 344 , and Met 346 constitute the pocket base. The substrate methionine side chain C⑀ and S␦ have close contacts with Leu 298 C ␦1 (3.3 Å), C ␦2 (3.2 Å), Tyr 344 C ␦2 (3.4 Å), Thr 290 C ␥2 (3.4 Å), and Phe 191 C (4.2 Å). In particular, Leu 298 , directly facing toward the substrate, may be important for the substrate specificity, because the D-aminoacylases with glutamate or aspartate preference contain an arginine residue at this position.
Ligand-mediated Conformational Switch-The D-aminoacylase structure here seems a closed conformation, because the active-site cavity is almost inaccessible to solvent (Fig. 5). The zinc ions lie in the deepest part of the active site, and the hydrophobic side chain is close to the opening of the pocket. The narrow opening of the cavity is capped by the 63-residue insertion. The 63-residue insertion borders the active site and contains many putative substrate-interacting residues as mentioned above. This domain may act as a gate controlling access to the active site, affecting both substrate access and product release. Particularly, the two antiparallel ␤-strands (residues 287-293 and 339 -346) may act as the fulcrum of the conformational change, because substrate-contacting residues are located in these regions. The closed conformation described here may be due to the interaction between the second acetate ACT2 and Tyr 283 and Ser 289 , sealing the entrance. Then the substrate binding would induce a closed conformation to sequester the reaction complex from solvent.
This type of conformational switch upon the substrate binding is also observed in other ␣/␤-barrel amidohydolases, including adenosine deaminase (18), cytosine deaminase (19), and urease (30,31). In these three enzymes, the conformational changes appear to be induced by direct contacts between protein and the bound substrate. In adenosine deaminase and cytosine deaminase, similar flaps are formed by the insertions between the ␤1-strand and the ␣1-helix. On the other hand, in urease, the lid is formed from the insertion between ␤7 and ␣7, as that in the D-aminoacylase. It is worth noting that the enzyme inhibition mechanism of D-aminoacylase by acetate may be similar to that of urease by ␤-ME, because in both cases, one inhibitor molecule bridges the metal ions and another molecule induces a closed conformation. In the ␤-MEinhibited urease, one ␤-ME ligates the two nickel ions, and another ␤-ME forms a mixed disulfide with Cys 322 sealing the entrance (30).
The Proposed Catalytic Mechanism-The strong structural homology of the ␣/␤-barrel amidohydrolases is also reflected in their catalytic mechanisms, in particular, preparation of the active nucleophile for the hydrolytic reaction is very similar (13)(14)(15)(16)(17)(18)(19)31). The ␣ metal ion functions in activation of the nucleophile water by lowering its pK a , while the ␤ metal ion serves as an electrophilic catalyst to polarize the carbonyloxygen bond of the substrate. The highly conserved Asp 366 probably acts as a general base to activate the nucleophile water. The proximity of His 67 N ␦1 to Asp 366 O ␦1 (3.2 Å) could facilitate the proton abstraction and donation, and the proximity of His 69 N ⑀2 to ACT1 O 1 (3.1 Å), and to Asp 366 O ␦1 (3.3 Å), might further assist in activating the attacking water molecule and stabilizing the negatively charged intermediate.
The crystal structure of D-aminoacylase with the modeled substrate provides the structural basis for the enzyme catalytic mechanism. Together with the similar mechanisms in the ␣/␤barrel amidohydrolases, we propose a catalytic mechanism for D-aminoacylase in Scheme 1. First, Asp 366 abstracts the proton from the water molecule, and the tightly bound zinc ion polarizes the carbonyl-oxygen bond, thus facilitating the nucleophilic attack on the amide carbon atom to form the tetrahedral intermediate. Cleavage of the carbon-nitrogen bond is assisted by the simultaneous protonation of the amide nitrogen. The newly formed acetate then ligates the zinc ion. An N-acetyl-Lmethionine substrate can also be modeled into the active site; however, for the L-isomer the interaction between the substrate amide and Ser 289 backbone carbonyl would be missing, perhaps resulting in lack of proper orientation of the amide carbon for water attacking, then with thus 100 times lower hydrolysis efficiency than the D-form substrate (2).
Conclusion-In summary, the crystal structure of D-aminoacylase reveals that the enzyme indeed belongs to the ␣/␤barrel amidohydrolase superfamily and defines a novel subset. A putative substrate-binding pocket with key residues is identified. The unusual 63-residue large insertion involves in the substrate specific recognition and the active-site entrance switch. On the basis of our structural information, some protein engineering trials such as deletion of the small ␤-domain and change of the substrate specificity by using mutagenesis are under investigation.
FIG. 5. The closed conformation of the D-aminoacylase. A, the protein surface colored by electrostatic potential from Ϫ40 k B T (red) to 40 k B T (blue) and shown without the insertion. The entrance to the active site is open as the 63-residue insertion is removed. The substrate N-acetyl-D-methionine and the tightly bound zinc ion are also shown as a stick model and a purple sphere in the active site pocket, respectively. B, the active site is closed with the presence of the insertion. The protein is shown as ribbons with the small ␤-domain, the ␣/␤-barrel, and the insertion colored blue, cyan, and green, respectively. The surfaces of the ␣/␤-barrel and the insertion are shown only for regions near the active site. The view of B is rotated 45 degrees about the vertical axis. SCHEME 1. The proposed catalytic mechanism for D-aminoacylase.