Crystal Structure of D-Aminoacylase from Alcaligenes faecalis DA1

A NOVEL SUBSET OF AMIDOHYDROLASES AND INSIGHTS INTO THE ENZYME MECHANISM*

Shwu-Huey LiawDagger §, Shen-Jia Chen§, Tzu-Ping Ko||, Cheng-Sheng Hsu§**, Chun-Jung ChenDagger Dagger , Andrew H.-J. Wang||§§, and Ying-Chieh Tsai§§§

From the Dagger  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 Dagger Dagger  X-ray Structural Biology Group, Synchrotron Radiation Research Center, Hsinchu 30077, Taiwan

Received for publication, October 22, 2002, and in revised form, November 22, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the hydrolysis of N-acyl-D-amino acids. We report here the first D-aminoacylase crystal structure from A. faecalis at 1.5-Å resolution. The protein comprises a small beta -barrel, and a catalytic (beta alpha )8-barrel with a 63-residue insertion. The enzyme structure shares significant similarity to the alpha /beta -barrel amidohydrolase superfamily, in which the beta -strands in both barrels superimpose well. Unexpectedly, the enzyme binds two zinc ions with widely different affinities, although only the tightly bound zinc ion is required for activity. One zinc ion is coordinated by Cys96, His220, and His250, while the other is loosely chelated by His67, His69, and Cys96. This is the first example of the metal ion coordination by a cysteine residue in the superfamily. Therefore, D-aminoacylase defines a novel subset and is a mononuclear zinc metalloenzyme but containing a binuclear active site. The preferred substrate was modeled into a hydrophobic pocket, revealing the substrate specificity and enzyme catalysis. The 63-residue insertion containing substrate-interacting residues may act as a gate controlling access to the active site, revealing that the substrate binding would induce a closed conformation to sequester the catalysis from solvent.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

N-Acyl-D-amino acid amidohydrolases (D-aminoacylases, EC 3.5.1.14) catalyze the zinc-assisted hydrolysis of N-acyl-D- amino 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 L-amino acids by optical resolution using L-aminoacylase immobilized on DEAE-Sephadex has been used in industry. Therefore, production of D-amino acids using D-aminoacylase has commercial importance.

Several D-aminoacylases screened from microorganisms in various soils have been isolated and characterized (1-6). Because of more thermostability, high substrate specificity with hydrophobic D-amino acids such as N-acetyl-D-methionine, and high affinity to DEAE resins, the D-aminoacylase from Alcaligenes faecalis DA1 is more suitable for optical resolution of N-acyl-DL-amino acids (2). The DA1 D-aminoacylase shares 40-80% sequence identity to those from A. xylosoxydans A-6, and Pyrococcus abyssi, but no significant homology with L-aminoacylases (7-10). Sequence homology search also revealed that the enzyme N-terminal segment (residues 8-96) shared significant similarity within a variety of amidohydrolases including urease (10). The structural fold was predicted to be similar to urease and dihydroorotase, which have grouped into a novel alpha /beta -barrel amidohydrolase superfamily (10, 11). And the metal ligands in D-aminoacylases have been proposed based on structural prediction (10) and mutational studies (10, 12).

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 (beta alpha )8-barrel with conserved metal ligands, four histidines and one aspartate, at the C-terminal ends of strands beta 1 (HXH), beta 5 (His), beta 6 (His), and beta 8 (Asp), and led to discovery of the alpha /beta -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.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
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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 P212121, with cell dimensions a = 60.2 Å, b = 76.6 Å, and c = 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 His250, Thr290, and Thr406 due to hydrogen bond interactions. Figs. 1, 3B, and 4 were generated by MOLSCRIPT (22), Fig. 2 by INSIGHT II (Molecular Simulation Inc.), Fig. 3A by BOBSCRIPT (23), and Fig. 5 by GRASP (24). The atomic coordinates and structure factors have been deposited in the Protein Data Bank (code 1M7J).

                              
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Table I
Statistics of data collection and structural refinement
Values in parentheses are for the highest resolution shell. The Rfree value is for a 5% test set (5,056 reflections).


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 beta -barrel (residues 7-61 and 425-480) and a catalytic alpha /beta -barrel (residues 62-414) (Fig. 1). There are two insertions in the alpha /beta -barrel: residues 147-165 between the beta 3 strand and alpha 3-helix, forming a helix and a random coil, and residues 285-347 between beta 7 and alpha 7, forming two helices and four beta  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 beta 2 and alpha 2 (residues 99-127) and between beta 8 and beta 8 (residues 366-398).


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Fig. 1.   The A. faecalis D-aminoacylase structure. A, ribbon diagram of the Calpha backbone. The structural fold contains a small beta -barrel comprising both the N and the C terminus (residues 7-61 and 425-480), a central alpha /beta -barrel (residues 62-414), with a 63-residue insertion (residues 285-347). The small beta -barrel may be responsible for the structural stabilization, the alpha /beta -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 alpha /beta -barrel. The tightly bound zinc ion is chelated by Cys96, His220, His250, and the first acetate.

Structural similarity search by DALI (25) revealed that the closest structural matches of D-aminoacylase are seven members of the recently identified alpha /beta -barrel amidohydrolase superfamily mentioned above (13-19), in particular, urease and cytosine deaminase, with r.m.s.1 deviations of 2.9 Å (325 Calpha atoms with 18% sequence identity) and 3.8 Å (330 Calpha atoms with 12% sequence identity), respectively. Structural comparison reveals that the beta -strands in both the small beta  barrel and the catalytic alpha /beta -barrel correspond closely, whereas the external helices and surface-exposed loops diverge significantly. Despite the apparent lack of sequence similarity, the eight beta -strands (56 structurally equivalent residues) of the alpha /beta -barrels overlay within 1.2-2.0 Å r.m.s. deviations (Fig. 2A). On the other hand, the size of the small beta -domain, comprising both the N and the C termini, varies greatly in different structures. The beta -strands of the beta -domains (48 structurally equivalent residues) overlay within 1.0-1.3 Å r.m.s. deviations (Fig. 2B). The small beta -barrel does not contribute any residues to the active site and appears to play a structural role.


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Fig. 2.   Structural superposition of D-aminoacylase (green), urease (red, Protein Data Bank code 1UBP), dihydroorotase (pink, Protein Data Bank code 1J79), dihydropyrimidinase (blue, Protein Data Bank code 1GKP), cytosine deaminase (yellow, Protein Data Bank code 1K6W), adenosine deaminase (orange, Protein Data Bank code 1A4M), and phosphotriesterase homology protein (black, Protein Data Bank code 1BF6), with a close view of the elliptically distorted alpha /beta -barrel (A) and the small beta -barrel (B). The two nickel ions in urease at the alpha  and beta  sites are shown as red spheres. The letters N and C indicate the N and C termini in D-aminoacylase. The beta -strands in both barrels superimpose quite well.

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 Cys96 Sgamma (2.24 Å), His220 Ndelta 1 (2.08 Å), His250 Nepsilon 2 (2.04 Å), and an acetate molecule, ACT1 O2 (2.04 Å), from the crystallization solution.


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Fig. 3.   The metal center. A, the 2Fo - Fc electron density map in the zinc center contoured at the 3sigma level and is shown in green, and the weak density in the 2Fo - Fc map for the loosely bound zinc ion contoured at the 2sigma 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 alpha  metal-binding site is more buried, while the beta  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 (Cys96) in D-aminoacylse, and the third conserved histidine (His214) in cytosine deaminase, compensate the missing carboxylated lysine.

There is another potential metal-binding site, surrounded by His67, His69, Cys96, Asp366, and ACT1, and similar to the metal-binding sites in the alpha /beta -barrel amidohydrolase superfamily (13-19). 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 g·atom per mole of enzyme (10). Therefore, the weak electron density is assigned as zinc, which is tetrahedrally ligated by His67 Ne2 (2.01 Å), His69 Nepsilon 2 (2.05 Å), Cys96 Sgamma (2.19 Å), and ACT1 O1 (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 Å2 for the loosely bound zinc ion and an occupancy of 1.0 with B-factor of 7.3 Å2 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 beta -lactamase from Bacillus cereus (26-28).

Even though beta -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 (Kd ~10 µM), whereas the B. cereus enzyme binds zinc ions with very distinct affinities (Kd ~1 µM 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-beta -lactamases (27, 28).

A Novel Subset of the alpha /beta -Barrel Amidohydrolase Superfamily-- To date, there are two subsets in the alpha /beta -barrel amidohydrolase superfamily based on the metal centers with four conserved histidines and one aspartate (Fig. 3B). In the binuclear subset (13-17), the more buried metal ion (alpha  site) is coordinated by the first two conserved histidines from the common zinc-binding HXH motif (29), the conserved aspartate, and two bridging ligands, whereas the more solvent-exposed metal ion (beta  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 alpha  site ligated by the first three conserved histidines and one water molecule.

In D-aminoacylase, one zinc ion binds strongly at the beta  site, and the other binds weakly at the alpha  site. This is the first example of a cysteine residue (Cys96) 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 Cys96 occupying the position of the carboxylated lysine (Fig. 3B). In cytosine deaminase and adenosine deaminase, the third conserved histidine compensates the missing carboxylated 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 beta 4 strand in the alpha /beta -barrel (15; Fig. 2A). The zinc-zinc distance of 3.1 Å in D-aminoacylase is similar to the nickel-nickel distance of 3.1 Å in the beta -mercaptoethanol (beta -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 (13-17, 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 beta -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). The first one (ACT1) ligating the zinc ion(s) may occupy the product acetate-binding site in the hydrolysis of N-acteyl-D-amino acid. The second one (ACT2) forms extensive interactions with Lys252 Nzeta (2.7 Å), Arg377 Neta 1 (2.7 Å), Neta 2 (3.0 Å), Tyr283 Oeta (2.8 Å), and Ser289 N (2.8 Å), suggesting that this acetate molecule occupies the binding position of the substrate carboxylate group. Around this region, there is a hydrophobic pocket formed by the side chains of Phe191, Tyr192, Lys252, Met254, Leu298, Tyr344, Met346, and Met347.


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Fig. 4.   The putative substrate-binding site. A, the two acetate-binding sites in the active site cavity (Act1 and Act2). Residues surrounded the binding site are displayed as ball-and-stick representations and the zinc ion as a purple sphere. B, the proposed substrate-binding site with the modeled N-acetyl-D-methionine in ball-and-stick and the active water molecule (Wat) as a green sphere. The oxygen atoms of the acetates and substrate are expected to bind at the same position because of the extensive interactions. The first acetate may displace the attacking water molecule at the metal center.

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 alpha /beta -barrel amidohydrolase superfamily members (13-19, 31), the amide oxygen atom coordinates the beta  ion (which is likely at the ACT1 O2 position), and the amide carbon atom must be in close proximity to the active water molecule (the ACT1 O1 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 Ser289 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 Thr290, Phe191, Lys252, Met254, and Met347, in which Leu298, Tyr344, and Met346 constitute the pocket base. The substrate methionine side chain Cepsilon and Sdelta have close contacts with Leu298 Cdelta 1 (3.3 Å), Cdelta 2 (3.2 Å), Tyr344 Cdelta 2 (3.4 Å), Thr290 Cgamma 2 (3.4 Å), and Phe191 Cxi (4.2 Å). In particular, Leu298, 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 beta -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 Tyr283 and Ser289, sealing the entrance. Then the substrate binding would induce a closed conformation to sequester the reaction complex from solvent.


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Fig. 5.   The closed conformation of the D-aminoacylase. A, the protein surface colored by electrostatic potential from -40 kBT (red) to 40 kBT (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 beta -domain, the alpha /beta -barrel, and the insertion colored blue, cyan, and green, respectively. The surfaces of the alpha /beta -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.

This type of conformational switch upon the substrate binding is also observed in other alpha /beta -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 beta 1-strand and the alpha 1-helix. On the other hand, in urease, the lid is formed from the insertion between beta 7 and alpha 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 beta -ME, because in both cases, one inhibitor molecule bridges the metal ions and another molecule induces a closed conformation. In the beta -ME-inhibited urease, one beta -ME ligates the two nickel ions, and another beta -ME forms a mixed disulfide with Cys322 sealing the entrance (30).

The Proposed Catalytic Mechanism-- The strong structural homology of the alpha /beta -barrel amidohydrolases is also reflected in their catalytic mechanisms, in particular, preparation of the active nucleophile for the hydrolytic reaction is very similar (13-19, 31). The alpha  metal ion functions in activation of the nucleophile water by lowering its pKa, while the beta  metal ion serves as an electrophilic catalyst to polarize the carbonyl-oxygen bond of the substrate. The highly conserved Asp366 probably acts as a general base to activate the nucleophile water. The proximity of His67 Ndelta 1 to Asp366 Odelta 1 (3.2 Å) could facilitate the proton abstraction and donation, and the proximity of His69 Nepsilon 2 to ACT1 O1 (3.1 Å), and to Asp366 Odelta 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 alpha /beta -barrel amidohydrolases, we propose a catalytic mechanism for D-aminoacylase in Scheme 1. First, Asp366 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-L-methionine substrate can also be modeled into the active site; however, for the L-isomer the interaction between the substrate amide and Ser289 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).


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Scheme 1.   The proposed catalytic mechanism for D-aminoacylase.

Conclusion-- In summary, the crystal structure of D-aminoacylase reveals that the enzyme indeed belongs to the alpha /beta -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 beta -domain and change of the substrate specificity by using mutagenesis are under investigation.

    ACKNOWLEDGEMENTS

The synchrotron radiation experiments were performed at the Synchrotron Radiation Research Center, Hsinchu, Taiwan, at the Photon Factory, Tsukuba, Japan, and at the SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (Proposal No. 2002A0504-CL1-np).

    FOOTNOTES

* This work was supported by National Science Council Grants NSC 91-2311-B-010-010, NSC 90-2321-B-002-002, and NSC 90-2321-B-001-015.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1M7J) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

§§ To whom correspondence may be addressed: Inst. of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan. E-mail: ahjwang@gate.sinica.edu.tw (for A. H.-J. W.) or Inst. of Biochemistry, National Yang-Ming University, Taipei 11221, Taiwan. Tel.: 886-2-2826-7278; Fax: 886-2-2820-2449; E-mail: shliaw@ym.edu.tw (for) Y.-C. T.).

Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M210795200

    ABBREVIATIONS

The abbreviations used are: r.m.s., root mean square; beta -ME, beta -mercaptoethanol; kBT, Boltzmann's constant × temperature (1.38 × 10-23 J/K × T = 1.38 × 10-23 J).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Yang, Y. B., Lin, C. S., Tseng, C. P., Wang, Y. J., and Tsai, Y. C. (1991) Appl. Environ. Microbiol. 57, 1259-1260
2. Tsai, Y. C., Lin, C. S., Tseng, T. H., Lee, H., and Wang, Y. J. (1992) Enzyme Microbial. Technol. 14, 384-389[CrossRef][Medline] [Order article via Infotrieve]
3. Yang, Y. B., Hsiao, K. M., Li, H., Yano, H., Tsugita, A., and Tsai, Y. C. (1992) Biosci. Biotechnol. Biochem. 56, 1392-1395[Medline] [Order article via Infotrieve]
4. Sugie, M., and Suzuki, H. (1978) Agric. Biol. Chem. 44, 107-113
5. Sakai, K., Obata, T., Ideta, K., and Moriguchi, M. (1991) J. Ferment. Bioeng. 71, 79-82[CrossRef]
6. Moriguchi, M., Sakai, K., Miyamoto, Y., and Wakayama, M. (1993) Biosci. Biotechnol. Biochem. 57, 1149-1152[Medline] [Order article via Infotrieve]
7. Wakayama, M., Ashika, T., Miyamoto, Y., Yoshikawa, T., Sonoda, Y., Sakai, K., and Moriguchi, M. (1995) J. Biochem. (Tokyo) 118, 204-209[Abstract]
8. Wakayama, M., Katsuno, Y., Hayashi, S., Miyamoto, Y., Sakai, K., and Moriguchi, M. (1995) Biosci. Biotechnol. Biochem. 59, 2115-2119[Medline] [Order article via Infotrieve]
9. Wakayama, M., Watanabe, E., Takenaka, Y., Miyamoto, Y., Tau, Y., Sakai, K., and Moriguchi, M. (1995) J. Ferment. Bioeng. 80, 311-317[CrossRef]
10. Hsu, C. S., Lai, W. L., Chang, W. W., Liaw, S. H., and Tsai, Y. C. (2002) Protein Sci. 11, 2545-2550[Abstract/Free Full Text]
11. Holm, L., and Sander, C. (1997) Proteins 28, 72-82[CrossRef][Medline] [Order article via Infotrieve]
12. Wakayama, M., Yada, H., Kanda, S., Hayashi, S., Yatsuda, Y., Sakai, K., and Moriguchi, M. (2000) Biosci. Biotechnol. Biochem. 64, 1-8[Medline] [Order article via Infotrieve]
13. Jabri, E., Carr, M. B., Hausinger, R. P., and Karplus, P. A. (1995) Science 268, 998-1004[Medline] [Order article via Infotrieve]
14. Benning, M. M., Shim, H., Raushel, F. M., and Holden, H. M. (2001) Biochemistry 40, 2712-2722[CrossRef][Medline] [Order article via Infotrieve]
15. Buchbinder, J. L., Stephenson, R. C., Dresser, M. J., Pitera, J. W., Scanlan, T. S., and Fletterick, R. J. (1998) Biochemistry 37, 5096-5106[CrossRef][Medline] [Order article via Infotrieve]
16. Thoden, J. B., Phillips, G. N., Neal, T. M., Raushel, F. M., and Holden, H. M. (2001) Biochemistry 40, 6989-6997[Medline] [Order article via Infotrieve]
17. Abendroth, J., Niefind, K., and Schomburg, D. (2002) J. Mol. Biol. 320, 143-156[CrossRef][Medline] [Order article via Infotrieve]
18. Wilson, D. K., Rudolph, F. B., and Quiocho, F. A. (1991) Science 252, 1278-1284[Medline] [Order article via Infotrieve]
19. Ireton, G. C., McDermott, G., Black, M. E., and Stoddard, B. L. (2002) J. Mol. Biol. 315, 687-697[CrossRef][Medline] [Order article via Infotrieve]
20. Hsu, C. S., Chen, S. J., Tsai, Y. C., Lin, T. W., Liaw, S. H., and Wang, A. H. J. (2002) Acta Crystallogr. Sect. D Biol. Crystallogr. 58, 1482-1483[Medline] [Order article via Infotrieve]
21. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
22. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
23. Esnouf, R. M. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 938-940[CrossRef][Medline] [Order article via Infotrieve]
24. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296[Medline] [Order article via Infotrieve]
25. Dietmann, S., Park, J., Notredame, C., Heger, A., Lappe, M., and Holm, L. (2001) Nucleic Acids Res. 29, 55-57[Abstract/Free Full Text]
26. Concha, N. O., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Struct. Fold. Des. 4, 823-836
27. Fabiane, S. M., Sohi, M. K., Wan, T., Payne, D. J., Bateson, J. H., Mitchell, T., and Sutton, B. J. (1998) Biochemistry 37, 12404-12411[CrossRef][Medline] [Order article via Infotrieve]
28. Chantalat, L., Duee, E., Galleni, M., Frere, J. M., and Dideberg, O. (2000) Protein Sci. 9, 1402-1406[Abstract]
29. Vallee, B. L., and Auld, D. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2715-2718[Abstract]
30. Benini, S., Rypniewski, W. R., Wilson, K. S., Ciurli, S., and Mangani, S. (1998) J. Biol. Inorg. Chem. 3, 268-273[CrossRef]
31. Benini, S., Rypniewski, W. R., Wilson, K. S., Miletti, S., Ciurli, S., and Mangani, S. (1999) Struct. Fold. Des. 7, 205-216[Medline] [Order article via Infotrieve]


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