JBC Transcription and Nuclear Factor Monoclonals

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Originally published In Press as doi:10.1074/jbc.M607270200 on September 8, 2006

J. Biol. Chem., Vol. 281, Issue 45, 34365-34373, November 10, 2006
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Crystal Structures of Nonoxidative Zinc-dependent 2,6-Dihydroxybenzoate ({gamma}-Resorcylate) Decarboxylase from Rhizobium sp. Strain MTP-10005*

Masaru Goto{ddagger}§, Hideyuki Hayashi{ddagger}, Ikuko Miyahara§, Ken Hirotsu§1, Masahiro Yoshida, and Tadao Oikawa||

From the {ddagger}Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan, §Department of Chemistry, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan, Department of Biotechnology, Faculty of Engineering, Kansai University, and the ||Kansai University High Technology Research Center, Osaka 564-8680, Japan

Received for publication, August 1, 2006 , and in revised form, September 1, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Reversible 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 belongs to a nonoxidative decarboxylase family. We have determined the structures of the following three forms of the enzyme: the native form, the complex with the true substrate (2,6-dihydroxybenzoate), and the complex with 2,3-dihydroxybenzaldehyde at 1.7-, 1.9-, and 1.7-Å resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of one ({alpha}beta)8 triose-phosphate isomerase-barrel domain with three functional linkers and one C-terminal tail. The native enzyme possesses one Zn2+ ion liganded by Glu8, His10, His164, Asp287, and a water molecule at the active site center, although the enzyme has been reported to require no cofactor for its catalysis. The substrate carboxylate takes the place of the water molecule and is coordinated to the Zn2+ ion. The 2-hydroxy group of the substrate is hydrogen-bonded to Asp287, which forms a triad together with His218 and Glu221 and is assumed to be the catalytic base. On the basis of the geometrical consideration, substrate specificity is uncovered, and the catalytic mechanism is proposed for the novel Zn2+-dependent decarboxylation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The nonoxidative decarboxylation catalyzed by decarboxylases such as 2,3-dihydroxybenzoate (1-5), 2,5-dihydroxybenzoate (6), 3,4-dihydroxybenzoate (7), 4,5-dihydroxyphthalate (8-10), and 4-hydroxybenzoate decarboxylase (11, 12) is a poorly understood reaction. These enzymes have been reported to require neither a cofactor such as NAD+, pyridoxal 5'-phosphate, or thiamine monophosphate nor a pyruvoyl group for catalytic activity. In studies on these enzymes, the interest is focused on their substrate specificities and catalytic mechanisms.

We isolated a thermophilic reversible 2,6-dihydroxybenzoate ({gamma}-resorcylate) decarboxylase (GRDC)2 from Rhizobium sp. strain MTP-10005 and characterized it (13). The GRDC catalyzes the decarboxylation of 2,6- and 2,3-dihydroxybenzoate to 1,3-dihydroxybenzene (resorcinol) and 1,2-dihydroxybenzene, respectively but does not act on 2,4-, 2,5-, 3,4-, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate (Scheme 1). 2,6-Dihydroxybenzoate is an important intermediate of medicine and agricultural or industrial chemicals (14-16). However, it is generated together with 2,4-dihydroxybenzoate as a by-product at a rate of about half and half by traditional chemical methods (17). 2,6-Dihydroxybenzoate is expected to be produced specifically from 2,6-dihydroxybenzene by the reverse carboxyl reaction of GRDC.

Recently, Ishii et al. (18) reported the purification and characterization of GRDC from Rhizobium radiobacter WU-0108, Agrobacterium tumefaciens IAM12048 (19), and Pandoraea sp. 12B-2 (20). They reported that these enzymes also catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate without cofactors and have a similar substrate specificity. Orotidine 5'-monophosphate decarboxylase catalyzes the cofactorindependent decarboxylation. On the basis of the x-ray structure of the enzyme, it is proposed that the decarboxylation of orotidine 5'-monophosphate proceeds by an electrophilic substitution mechanism in which decarboxylation and carbon-carbon bond protonation by Lys62 occur in a concerted way (21, 22).

To elucidate the overall and active-site structure, the substrate recognition, and the reaction mechanism, we have determined the crystal structures of GRDC from the Rhizobium sp. strain MTP-10005 in the native form, GRDC complexed with the substrate 2,6-dihydroxybenzoate, and GRDC complexed with substrate analogue 2,3-dihydroxybezaldehyde at 1.7-, 1.9-, and 1.7-Å resolutions, respectively. Interestingly, these structures revealed that Zn2+ ion is bound to the active center to catalyze the reaction. We now report the first structure of GRDC as a novel Zn2+-dependent reversible decarboxylase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Crystallization and Data Collection—The purification of the enzyme has been reported elsewhere (13). The initial screening for the crystallization conditions was performed using the sparse matrix screens (Crystal Screens I and II) from Hampton Research (23). Crystallization of the native GRDC was carried out at 293 K by the hanging drop vapor diffusion method, using 3.6 mg/ml protein solution and a reservoir solution containing 15% (w/v) PEG8000, 5% (v/v) ethylene glycol, 10% (v/v) glycerol, 100 mM Tris-HCl, pH 7.5. The native crystal was obtained from a drop composed of a 1:1 volume ratio of the reservoir solution and the protein solution. Within 2 weeks, crystals had grown to dimensions of ~0.05 x 0.15 x 0.40 mm3. Crystals of GRDC·2,6-dihydroxybenzoate and GRDC·2,3-dihydroxybenzaldehyde were obtained at 293 K by soaking the native crystals in a reservoir solution containing 40 mM 2,6-dihydroxybenzoate for 10 min and 40 mM 2,3-dihydroxybenzaldehyde for 40 min, respectively, before data collection.


Figure 1
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  		SCHEME 1
 
The x-ray diffraction data set for the native crystal was collected to 1.7 Å resolution at 100 K using a wavelength of 1.00 Å on the NW12 station equipped with an ADSC Quantum 4R CCD detector system at the photon factory, KEK (Tsukuba, Japan). The x-ray data sets for the GRDC·2,6-dihydroxybenzoate crystal and the GRDC·2,3-dihydroxybenzaldehyde crystal were collected to 1.9 and 1.7 Å resolution at 100 K using a wavelength of 1.0 Å on the BL41XU station and a ADSC Quantum 315 CCD detector at the SPring-8 (Hyogo, Japan). These three crystals are isomorphous with a space group of P212121 and have average cell dimensions of a = 109.2, b = 113.66, and c = 119.2 Å. There are four subunits in the asymmetric unit, and ~44% of the crystal volume is occupied by solvent (24). The heavy atom derivatives were obtained by soaking unliganded crystals in solutions containing heavy atom reagents. The data sets for heavy atom derivatives were collected to 2.15 and 2.45 Å resolution at 100 K on the NW12 station at the photon factory (KEK). All the data were processed and scaled using the program HKL2000 (25) (Table 1).


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  		TABLE 1
Data collection and refinement statistics

 
Structure Determination and Refinement—The structure of the native GRDC was solved by the multiple isomorphous replacement with anomalous scattering method, using two isomorphous data sets. The scaling of all the data and the map calculations were performed with the CCP4 program suite (26). The positions of one mercury and five platinum sites were determined with the program SOLVE (27). The resulting isomorphous replacement with anomalous scattering map has a mean figure-of-merit of 0.335 at a resolution of 20-3.0 Å. The map was significantly improved by the process of solvent flattening and local symmetry averaging with the program RESOLVE (28). The mean figure-of-merit reached 0.712 with the same resolution range. The map was of good quality, and the model of the subunit was gradually built into the 3.0 Å map through several cycles of model building using the program O (29). The model was refined by simulated annealing and energy minimization with the program CNS (30). A random sample with 10% of the reflections was set apart for the calculation of the Rfree. Solvent molecules were picked up on the basis of the peak heights (3.0 {sigma}) and distance criteria (4.0 Å from protein or solvent) from the sigmaA-weighted Fo - Fc map. The procedure converged to Rfactor and Rfree values of 0.180 and 0.200, respectively, calculated for 163,233 reflections observed in a 40.3-1.70 Å resolution range (Table 1).

The same refinement procedure was applied to GRDC·2,6-dihydroxybenzoate and GRDC·2,3-dihydroxybenzaldehyde but using the coordinates of the unliganded enzyme as the initial model. When the Rfactor value became less than 30%, the simulated annealing 2Fo - Fc map showed residual electron densities corresponding to the bound 2,6-dihydroxybenzoate and 2,3-dihydroxybenzaldehyde. Solvent molecules were picked up from the sigmaA-weighted Fo - Fc map. Further model building and refinement cycles gave Rfactor values of 0.176 and 0.184 and Rfree values of 0.206 and 0.208 for GRDC·2,6-dihydroxybenzoate and GRDC·2,3-dihydroxybenzaldehyde, respectively (Table 1).

Quality of the Structure—The final model of the native GRDC comprises 325 residues (1-325) of 327 residues for subunit A, 324 residues (1-324) for subunit B, 323 residues (1-20 and 24-326) for subunit C, 321 residues (1-20 and 24-324) for subunit D, and four Zn2+ ions with 1223 water molecules. The average thermal factors of the main-chain atoms (N, C{alpha},C,and O) in subunits A, B, C, and D are 16.7, 16.8, 19.0, and 19.4 Å2, respectively. The final model of the GRDC·2,6-dihydroxybenzoate comprises 325 residues (1-325) of 327 residues for subunit A, 324 residues (1-324) for subunit B, 324 residues (1-20 and 23-326) for subunit C, 321 residues (1-20 and 24-324) for subunit D, four Zn2+ ions, and four 2,6-dihydroxybenzoates with 1034 water molecules. The average thermal factors of the main-chain atoms in subunits A, B, C, and D are 17.5, 17.5, 19.4, and 19.8 Å2, respectively. The final model of the GRDC·2,3-dihydroxybenzaldehyde comprises 325 residues (1-325) of 327 residues for subunit A, 324 residues (1-324) for subunit B, 323 residues (1-20 and 24-326) for subunit C, 321 residues (1-20 and 24-324) for subunit D, four Zn2+ ions, and four 2,3-dihydroxybenzaldehydes with 1223 water molecules. The average thermal factors of the main-chain atoms in subunits A, B, C, and D are 19.3, 19.3, 21.4, and 22.7 Å2, respectively. The differences in the average thermal factors between the subunits AB and CD in the final models are because of the asymmetry of the intermolecular interactions. The thermal factors for both complexes imply that crystal packing has less effect on subunits A and B than on subunits C and D.

The final models were validated with the PROCHECK (31) and CNS (30) programs. 99.4% of the amino acid residues in the final model were located in the most favorable and additionally allowed regions of the Ramachandran plot (Table 1). We note that one residue in the active site, Asp27, is located in the disallowed region in subunits A and B of the native GRDC. It is confirmed that the conformation of Asp27 is correct on the basis of the electron density map. We use subunit A and subunit B, if necessary, as the reference standards for the native and complex forms of GRDC. Structure diagrams were drawn using the programs MOLSCRIPT (32), RASTER3D (33), and POV-SCRIPT+ (34).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Overall and Subunit Structure—The overall structure of GRDC·2,6-dihydroxybenzoate is shown in Fig. 1A. The molecular mass of the enzyme is about 151 kDa by gel filtration and that of the subunit is 37.5 kDa by SDS-PAGE, suggesting that the GRDC exists as a homotetramer (13). The polypeptide chain is folded into a tetrameric form with a noncrystallographic 222 axis, having approximate dimensions of 95 x 85 x 50 Å. One subunit in the tetramer interacts with the other three subunits, and the surface areas of the subunit interfaces are 3431 Å2 for subunits A and B, 546 Å2 for subunits A and C, and 206 Å2 for subunits A and D. The largest of these areas is found between the subunits A and B (or C and D) indicating that the tetramer may be considered to be an assembly of two dimer units (a dimer of dimers) around a 2-fold axis.

The subunit structure of GRDC·2,6-dihydroxybenzoate with secondary structure assignments by the program DSSP (35) is shown in Fig. 1, B and C. The subunit C{alpha} carbon atoms except for Ala21-Asp28 in the native GRDC can be superimposed onto the corresponding ones in GRDC·2,6-dihydroxybenzoate and GRDC·2,3-dihydroxybenzaldehyde within root mean square deviations of 0.08 and 0.05 Å with the maximum displacement of 0.25 and 0.23 Å, respectively. Thus, the overall structure of the native GRDC is quite similar to that of the complex with the substrate or analogue when Ala21-Asp28 is neglected. However, the region of Ala21-Asp28 is mobile and plays an important role in substrate recognition (see "Induced Fit of Linker 1").

The subunit is divided into a ({alpha}beta)8 barrel, three linkers (linker 1, Glu9-Asp38; linker 2, Arg166-Thr86; and linker 3, His232-Glu255), and a C-terminal tail (Ser304-C-terminal). The barrel has a typical TIM-barrel structure (beta-strand S1, {alpha}-helix H3, S3, H4, S4, H5, S5, H6, S6, H7, S7, H8, S8, H10, S9, and H11) (Fig. 1, B and C). The linker 1 is inserted between S1 and H3 forming S2, H1, and H2. The random coiled linker 2 connects S6 and H7. The linker 3, composed of a random coil followed by H9, is between H8 and S8. The linkers play an important role in the formation of the dimeric unit because the residues from the linkers contribute to 46.5% of accessible surface area in the interface between subunits A and B. The C-terminal tail located at the dimer-dimer interface forms 40.5 and 46.6% of the interfaces between subunits A and C and subunits A and F, respectively, serving to form the tetramer.

The amino acid sequence of GRDC displays no significant homology to those of the known decarboxylases requiring a cofactor such as pyridoxal 5'-phosphate, thiamine pyrophosphate, and a pyruvoyl group. The program DALI (36) was used to search the Protein Data Bank data base for proteins possessing three-dimensional structures similar to that of the native GRDC. The highest Z scores (strength of structural similarity) were calculated to be 16.6 for the catalytic domain of D-hydantoinase (14% sequence identity; Protein Data Bank ID, 1YNY (37)), 16.3 for dihydroorotase (16%, 1J79 (38)), 16.1 for the catalytic domain of isoaspartyl dipeptidase (9%, 1PO9 (39)), 15.9 for renal dipeptidase (13%, 1ITQ (40)), 14.9 for phosphotriesterase (15%, 1PSC (41)), 14.0 for the catalytic domain of cytosine deaminase (11%, 1K6W (42)), 13.6 for the catalytic domain of n-acetylglucosamine-6-phosphate deacetylase (9%, 1UN7 (43)), 13.2 for the {alpha}-subunit of urease (13%, 1UBP (44)), 13.1 for D-aminoacylase (9%, 1M7J (45)), and 13.1 for adenosine deaminase (12%, 1A4M (46)). Because these enzymes are members of the recently identified TIM-barrel metallo-dependent hydrolase superfamily, GRDC is classified as a member of this superfamily. The superfamily is mainly divided into two subgroups depending on a bi-metal or mono-metal enzyme (47, 48), and GRDC thus belongs to the mono-metal-dependent hydrolase subgroup together with Fe3+-dependent cytosine deaminase (42) and Zn2+-dependent adenosine deaminase (46). Possibly, the active site structure of GRDC is more similar to that of adenosine deaminase than cytosine deaminase in terms of the mono-Zn2+ binding.


Figure 2
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  		FIGURE 1.
Ribbon structural drawings of GRDC complexed with 2,6-dihydroxybenzoate. A, GRDC tetramer viewed down the noncrystallographic 2-fold axis. Subunits A, B, C, and D are represented by green, yellow, red, and blue ribbons, respectively. The bound substrate 2,6-dihydroxybenzoate is shown by the Corey-Pauling-Koltun model. B, stereoview of the subunit with secondary structure assignments. ({alpha}beta)8 barrel, linkers 1-3, C-terminal tail, and linker 3 from the other subunit are shown in green, red, red, and blue ribbons, respectively. The 2,6-dihydroxybenzoate located at the active site is shown in yellow by the Corey-Pauling-Koltun model. {alpha}-Helices are denoted by H1-H13 and beta-strands by S1-S9. The N and C termini are labeled. C, side-view of the subunit with secondary structure assignments. The image structure is turned 90° clockwise around the horizontal axis relative to the image in B. The linker 1 in the native form (cyan) is superimposed.

 


Figure 3
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  		FIGURE 2.
Stereoview of the active site structure in GRDC. The residues from one subunit and the other subunit of the dimeric unit are shown by stick models in gray and orange, respectively. Zn2+ ion is drawn as a dark gray circle. The coordinate bonds between the Zn2+ ion and ligands are shown by dotted lines in magenta. A, active site of the native form. Eight water molecules are drawn as cyan circles. B, active site of GRDC·2,6-dihydroxybenzoate. The substrate, 2,6-dihydroxybenzoate, is shown by stick models in green. The electron density map contoured at 1.0 {sigma} is calculated using the data up to 1.90 Å resolution. Five water molecules are drawn as cyan circles. C, active site of GRDC·2,3-dihydroxybenzaldehyde. The substrate (2,6-dihydroxybenzoate) is superimposed onto 2,3-dihydroxybenzaldehyde in the GRDC·2,3-dihydroxybenzaldehyde complex. The substrate and 2,3-dihydroxybenzaldehyde are shown by stick models in green and magenta.

 
Induced Fit of Linker 1—One of the interesting features of GRDC is the insertion loop's conformational change to close the active site upon binding of the substrate (Fig. 1C). The C{alpha} atoms of 314 residues (1-19 and 31-325) of 325 residues in the native form can be superimposed onto the corresponding ones in GRDC·2,6-dihydroxybenzoate within 0.5 Å with a root mean square deviation of 0.08 Å. However, the root mean square deviation for 11 residues excluded from the calculation is 1.36 Å with a maximum displacement of 2.48 Å. This indicates that, upon binding of the substrate (2,6-dihydroxybenzoate), a part (Ser20-Ala30) of the linker 1 changes its main-chain conformation to close the active site from the solvent region. The bound substrate is almost isolated from the solvent region because accessible surface area of the substrate is reduced from 298 to 1 Å2 (Fig. 1A). This is also the case for the GRDC·2,3-dihydroxybenzaldehyde with the reduced accessible surface area of 3 Å2. The linker 1 acts as an active site lid and plays important roles in the substrate recognition and catalytic actions of the enzyme. It has been reported that isoaspartyl dipeptidase belonging to the hydrolase superfamily displays a conformational change similar to that of GRDC (39).

Zinc Coordination and Active Site Structure—When the Rfactor value became less than 30%, the simulated annealing 2Fo - Fc map showed a marked electron density peak located within 2.0-2.3 Å from Glu8, His10, His218, and Asp287. The peak was identified as that of Zn2+ ion by an anomalous scattering experiment, although Zn2+ ion was not contained in the buffer solution for purification or crystallization, and GRDC has been reported to require no cofactor. Zn2+ absorption edge was determined to be 1.2805 Å by x-ray absorption experiment. An electron density was observed at the position on Bijvoet difference map based on the data at {lambda} = 1.0000 Å, whereas there was no significant electron density on that based on the data at {lambda} = 1.2820 Å. We also determined the Zn2+ concentration by atomic absorption spectroscopy. Zinc atomic absorbencies followed at a wavelength of 213.9 nm, using a Hitachi Z-8200 model. The calculated number of Zn2+ ion per each tetramer was 4.01.

Active site components and bound Zn2+ ion in the native GRDC are shown in Figs. 2A and 3A. The Zn2+ ion located at the bottom of the active site is coordinated by the N-3 nitrogens of His10 (2.2 Å) and His164 (2.2 Å), the carboxylate oxygen of Glu8 (2.1 Å) and Asp287 (2.2 Å), and a water molecule (2.2 Å) with trigonal bipyramidal geometry. His164 and Asp287 act as axial ligands (aligned 173° apart through the Zn2+ ion) and His10, Glu8, and the bound water molecule form the vertices of three equatorial ligands. The side chains of Glu8, His10, His164, and Asp287 are arranged in geometrical positions favorable to act as ligands for Zn2+ by interactions with the neighboring residues of the active site. Glu8, His10, and His164 interact with Tyr162, Ser58, and Asn128, respectively. The carboxylate oxygen of Asp287 is hydrogen-bonded to the N-1 atom of His218, and the N-3 atom of His218 is further hydrogen-bonded to the carboxylate of Glu221, implying that the Asp:His:Glu triad forms the charge relay system. The Asp287 and His218 dyad is conserved in adenosine and cytosine deaminases, but it has been observed in the disallowed region of the Ramachandran plot (42, 46). The Zinc coordination scheme is very similar to those observed in adenosine deaminase and cytosine deaminase, although Glu8 in GRDC is replaced by a His residue in both deaminases.

The active site cavity, filled with eight water molecules, resides at the C-terminal side groove of the ({alpha}beta)8 barrel. The bottom of the cavity is composed of Zn2+ ion and the surrounding residues, and the active site wall is encircled by the C-terminal residues of the barrel (Ala61, Pro165, Pro185, Phe189, and Phe290), the linker 1 residues (Ser20 and Phe23), and the linker 3 residues (Asn234* (* indicates a residue from another subunit of the dimer unit) and Arg229*).

Binding Mode of the Substrate and Substrate Analogue—2,6-Dihydroxybenzoate is a true substrate for GRDC, and the GRDC·2,6-dihydroxybenzoate crystal was obtained by soaking the native crystal in a solution supplemented with 2,6-dihydroxybenzoate for 10 min. The stereo structure of GRDC·2,6-dihydroxybenzoate and the hydrogen-bonding scheme are shown in Figs. 2B and 3B, respectively. Upon binding of the substrate, three water molecules are expelled from the cavity. Concurrently, the linker 1 changes not only its main-chain but also its side-chain conformation to enclose the substrate in the cavity. The linker 1 residues, Ser20 and Phe23, behave like a lid for the active site cavity. Phe23 changes its side-chain direction toward the active site, and its side-chain phenyl group makes a van der Waals contact with the bound substrate with the shortest contact of 3.5 Å. The active site residues except for the linker 1 do not change their main-chain and side-chain conformations and retain the interactions among them upon binding of the substrate.

The substrate carboxylate is coplanar with the substrate phenyl ring. One side of the substrate phenyl ring plane is perpendicular to the phenyl ring of Phe189 with the shortest contact of 3.8 Å. The other side is parallel to Phe290 with {pi}-{pi} interaction within 3.5 Å. One of the carboxylate oxygen atoms of the substrate is directly coordinated to Zn2+ ion with a distance of 2.0 Å. The other oxygen atom forms a hydrogen bond with a water molecule. The substrate 2-hydroxy group is hydrogen-bonded to the carboxylate oxygen of Asp287, which is a ligand for Zn2+ ion and to two water molecules (W1 and W2), which are further hydrogen-bonded to Arg229*. Asp287 forms a triad together with His218 and Glu221. The 6-hydroxy group is hydrogen-bonded to a water molecule, which interacts with Ser20 and Ala61.

The activity of GRDC is inhibited by 2,3-dihydroxybenzaldehyde, which is an analogue of the substrate 2,3-dihydroxybenzoate. The stereo structure of GRDC·2,3-dihydroxybenzaldehyde and the hydrogen-bonding scheme are shown in Figs. 2C and 3C, respectively. The GRDC·2,3-dihydroxybenzaldehyde crystal was generated by the soaking method. The side-chain arrangements of the active site residues are quite similar to those observed in GRDC·2,6-dihydroxybenzoate. The location of 2,3-dihydroxybenzaldehyde is roughly the same as that of 2,6-dihydroxybenzoate, although the phenyl ring of 2,3-dihydroxybenzaldehyde rotates by about 30° around the axis perpendicular to the ring at the C-1 atom (Fig. 3C). The aldehyde carbonyl group in cis-position with respect to the C-1—C6 bond is coordinated to a Zn2+ ion. This might cause the phenyl ring rotation of 2,3-dihydroxybenzaldehyde compared with 2,6-dihydroxybenzoate. Asn234* is hydrogen-bonded to the 2- and 3-hydroxy groups. Arg229* and Asp287 interact with the 2-hydroxy group in the medium of two water molecules, W1 and W2.


Figure 4
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  		FIGURE 3.
Schematic diagram showing hydrogen bond and coordinate bond interactions. Putative hydrogen bond interactions are shown by dotted lines if the acceptor and donor are less than 3.5 Å apart. Coordinate bonds between Zn2+ ion and the active site residues are shown by solid lines if Zn2+ ion and the ligand are less than 2.2 Å apart. A, GRCD in the native form. B, GRCD·2,6-dihydroxybenzoate complex. C, GRCD·2,3-dihydroxybenzaldehyde complex.

 


Figure 5
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  		FIGURE 4.
Proposed reaction mechanism for decarboxylation in 2,6-dihydroxybenzoate decarboxylase.

 
GRDC catalyzes the decarboxylation of both 2,6-dihydroxybenzoate and 2,3-dihydroxybenzoate but does not act on 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate. This suggests that to catalyze the decarboxylation, it is necessary that the carboxylate and two hydroxy groups occupy three consecutive positions of the benzene ring (13). The substrate specificity shown by GRDC might be explained by the binding mode of 2,6-dihydroxybenzoate and 2,3-dihydroxybenzaldehyde to the active site (Figs. 2 and 3). The substrate carboxylate is coordinated to bind Zn2+ ion with the 3-position of the benzene ring directed toward the entrance of the cavity. Pro185, Phe189, and Phe290 limit the substrate to having a planar shape. Asp287, Arg229*, and Asn234* form a hydrophilic cluster together with water molecules to interact with the 2-hydroxy group or the 2- and 3-hydroxy groups. Ser20 acts as the recognition site for the 6-hydroxy group with the aid of a water molecule. These results show that the active site can recognize the hydroxy groups at positions 2, 3, and 6 of a benzene ring. When 3,4- or 3,5-dihydroxybenzoate is superimposed onto 2,6-dihydroxybenzoate in GRDC·2,6-dihydroxybenzoate, the hydrophilic 4- or 5-hydroxy group makes short contacts of 2-3 Å with the hydrophobic phenyl ring of Phe23 as a lid for the active site cavity. The lack of activity for decarboxylation of 2,4-, 2,5-, or 3,5-dihydroxybenzoate is attributable at least in part to the unfavorable access between the 4- or 5-hydroxy group and Phe23.

Mechanism—The crystallographic study revealed that GRDC is Zn2+-dependent decarboxylase, suggesting that the reaction mechanism is different from that of decarboxylase dependent on thiamine diphosphate (49, 50), pyridoxal 5'-phospahte (51), NAD (52, 53), NADP (54), FMN (55), or a pyruvoyl group (56, 57). The proposed mechanism of GRDC based on the geometrical consideration is shown in Fig. 4. Upon access of the substrate 2,6-dihydroxybenzoate to the active site cavity, GRDC changes the linker 1 conformation to enclose the substrate in the cavity. One of the substrate carboxyl oxygen atoms interacts with Zn2+ ion, and the substrate 2-hydoxyl group is hydrogen-bonded to the carboxylate of Asp287. Asp287 is liganded to Zn2+ ion and is involved in a triad together with His218 and Glu221. This indicates that Asp287 plays an important role in the catalysis. The water molecule (W1) interacts with positively charged His218 and Arg229* and the substrate 2-hydroxy group. The water molecule (W2), which interacts with Arg229*, bridges Asp287 and the substrate 2-hydroxy group by hydrogen bonds. Zn2+ ion, the triad (Asp287, His218, and Glu221), W1, and W2 form the specific recognition site for the carboxylate and the adjacent 2-hydroxy group of the substrate.

The negative charge on the substrate carboxylate group migrates via Zn2+ ion to the carboxylate of Asp287, which is polarized by the His218 and Glu221 pair (Fig. 4A). This increases the basicity of the carboxylate of Asp287, which then acts as a catalytic base to abstract a proton from the substrate 2-hydroxy group. The negative charge generated on the substrate O-2 flows into the aromatic ring and accumulates on the C-1 atom to form the substrate carbanion (Fig. 4B). The next step is the addition of a proton to the C-1 atom. The water molecule W1 interacts with positively charged Agr229* and His218, thereby reducing its pKa value. The pKa value would be further reduced after the 2-hydoxy proton abstraction by Asp287; the protonation at Asp287 weakens the hydrogen bond between Asp287 and His218 and decreases the electron density at N-1 of His218, thereby reducing the interaction between W1 and His218. W1, which is at a distance of 3.76 Å from the C-1 atom in Fig. 4A, approaches the C-1 atom within the distance of 3.4 Å on the formation of the carbanion followed by the adoption of sp3 hybridization by the C-1 atom. W1 is thus reasonably assumed to be the conjugate acid to shuttle its proton to the C-1 atom (Fig. 4C). Throughout this process, the repulsive interaction between the substrate carboxylate and the C-1 carbanion is suppressed by the coordination of the carboxylate to Zn2+ ion. The substrate C-2 carbonyl group then abstracts a proton from the carboxylate of Asp287 in concert with the decarboxylation of the substrate to yield carbon dioxide and resorcinol (Fig. 4, C and D).

The enzyme also catalyzes the decarboxylation of 2,3-dihydroxybenzoate. 2,3-Dihydroxybenzoate is modeled into the active site of GRDC·2,6-dihydroxybenzoate on the assumption that 2-hydroxybenzoate moieties of both substrates occupy the same position. The 3-hydroxy group of 2,3-dihydroxybenzoate is located at a position favorable for forming a hydrogen bond with Asn234* (Fig. 2C). Therefore, 2,3-dihydroxybenzoate is a good substrate for GRDC. After submission of this paper, we learned that the native structure of mono-metal-dependent decarboxylase ({alpha}-amino-beta-carboxymuconate-{epsilon}-semialdehyde decarboxylase) belonging to the TIM-barrel metallo-dependent hydrolase superfamily has been published (58).

In conclusion, we have determined the three-dimentional structures of the novel decarboxylase, GRDC, in the native form, complexed with substrate 2,6-dihydroxybenzoate and complexed with 2,3-dihydroxybenzaldehyde. This is the first structure of a Zn2+-dependent decarboxylase complex with the substrate or substrate analog belonging to the hydrolase superfamily. The arrangements of the active site residues, the substrate, the substrate analogue, and the interactions among them have been elucidated to provide a putative mechanism for catalysis of the enzyme. Asp287 and Zn2+ ion are identified as the catalyst.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2DVT, 2DVU, and 2DVX) 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 by the National Project on Protein Structural and Functional Analyses and in part by research grants from the Japan Foundation of Applied Enzymology and the Kansai University High Technology Research Center. 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.: 81-6-6605-3129; Fax: 81-6-6605-2557; E-mail: hirotsu{at}sci.osaka-cu.ac.jp.

2 The abbreviations used are: GRDC, 2,6-dihydroxybenzoate ({gamma}-resorcylate) decarboxylase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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M. Yoshida, T. Oikawa, H. Obata, K. Abe, H. Mihara, and N. Esaki
Biochemical and Genetic Analysis of the {gamma}-Resorcylate (2,6-Dihydroxybenzoate) Catabolic Pathway in Rhizobium sp. Strain MTP-10005: Identification and Functional Analysis of Its Gene Cluster
J. Bacteriol., March 1, 2007; 189(5): 1573 - 1581.
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