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Maleylacetate reductase (EC 220.127.116.11), which catalyzes the reduction of maleylacetate to 3-oxoadipate, plays an important role in the aerobic microbial catabolism of resorcinol. The enzyme has been crystallized at 293 K by the sitting-drop vapour-diffusion method supplemented with a microseeding technique, using ammonium sulfate as the precipitating agent. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 56.85, b = 121.13, c = 94.09 Å, β = 101.48°, and contained one dimeric molecule in the asymmetric unit. It diffracted to 1.79 Å resolution.
Rhizobium is a genus of tubercle-forming bacteria that grow in the root of a plant in symbiosis with other bacteria, such as Astragalus sinicus, Trifolium repens L. and Medicago polymorpha Linn, to fix nitrogen from the air (Beringer et al., 1979 ). Therefore, much attention has been paid to the Rhizobium genes and gene products owing to their role in the regulation of symbiosis. Despite extensive studies on Rhizobium (Wardhan et al., 1989 ; Krishnan & Pueppke, 1991 ; Harrison et al., 2003 ), there is still little information available on the molecular structure, function and detailed properties of the enzymes involved in its metabolic pathways.
In the course of a screening experiment, Rhizobium sp. strain MTP-10005 was isolated from natural river water as a microorganism with a high level of γ-resorcylate (2,6-hydroxybenzoate) decarboxylase (EC 4.1.1.x) activity (Yoshida et al., 2004 ). During the cloning of the graF gene encoding the enzyme γ-resorcylate decarboxylase, the genes graA, graB and graC were found immediately upstream and downstream of graF. They were equivalent to the genes encoding the enzymes in the resorcinol catabolic pathway (Yoshida et al., 2004 ) that is known in Pseudomonas putida (Chapman & Ribbons, 1976 ) and Trichosporon cutaneum (Gaal & Neujahr, 1979 ) but not in other Rhizobium sp. bacteria. Recently, four further genes (graD, graR, graE and graK) in the same pathway have been found (Yoshida et al., 2007 ). Thus, the gene cluster graRDAFCBEK has been identified as being involved in γ-resorcylate catabolism by Rhizobium sp. strain MTP-10005. Enzymological studies showed that graD, graA, graB and graC encode the reductase (GraD) and oxidase (GraA) components of resorcinol hydroxylase (EC 1.14.13.x), hydroxyquinol 1,2-dioxygenase (GraB; EC 18.104.22.168) and maleylacetate reductase (GraC; EC 22.214.171.124), respectively. In the aerobic bacterium Rhizobium sp. strain MTP-10005, γ-resorcylate is first converted to resorcinol by γ-resorcylate decarboxylase (GraF). Subsequently, resorcinol is sequentially converted into 3-oxoadipate via hydroxyquinol and maleylacetate by resorcinol hydroxylase (GraA and GraD), hydroxyquinol 1,2-dioxygenase (GraB) and maleylacetate reductase (GraC).
Recently, the crystal structures of 2,6-dihydroxybenzoate decarboxylase from Rhizobium sp. strain MTP-10005 were determined in three forms: in the native form, in complex with the native substrate 2,6-dihydroxybenzoate and in complex with 2,3-dihydroxybenzaldehyde (Goto et al., 2006 ). Moreover, a catalytic mechanism of 2,6-dihydroxybenzoate decarboxylase involving a novel Zn2+-dependent decarboxylation has been proposed based on these structures. γ-Resorcylate decarboxylation is the reaction that converts 2,6-dihydroxybenzoate to resorcinol just before the resorcinol catabolic pathway. We have been performing X-ray structural studies of the enzymes, including maleylacetate reductuse (GraC), that are involved in the resorcinol catabolism of Rhizobium sp. strain MTP-10005. Despite intensive biological investigations, no crystal structures of maleylacetate reductases from any species have yet been elucidated.
Maleylacetate reductases play a crucial role in the aerobic microbial degradation of aromatic compounds. In fungi and yeasts, the enzymes are involved in the catabolism of very common compounds such as phenol, tyrosine, benzoate, 4-hydroxybenzoate and resorcinol (Karasevich & Ivoilov, 1977 ; Buswell & Eriksson, 1979 ; Gaal & Neujahr, 1979 ; Sparnins et al., 1979 ; Anderson & Dagley, 1980 ; Jones et al., 1995 ). In bacteria, the enzymes contribute to the degradation of resorcinol, 2,4-dihydroxybenzoate (β-resorcylate) and 2,6-dihydroxybenzoate (γ-resorcylate) via hydroxyquinol and maleylacetate (Larway & Evans, 1965 ; Chapman & Ribbons, 1976 ; Stolz & Knackmuss, 1993 ; Yoshida et al., 2004 , 2007 ). Maleylacetate reductases catalyze NADH- or NADPH-dependent reduction, at the carbon–carbon double bond, of maleylacetate or 2-chloromaleylacetate to 3-oxoadipate or of substituted maleylacetates to substituted 3-oxoadipates. The enzymes that use 2-chloromaleylacetate as a substrate initially catalyze the NADH- or NADPH-dependent dechlorination of that compound to maleylacetate, which is subsequently reduced to 3-oxoadipate.
Maleylacetate reductase from Rhizobium sp. strain MTP-10005 catalyzes the reduction of maleylacetate to 3-oxoadipate and plays an important role in the catabolic pathway of the resorcinol produced through decarboxylation of 2,6-dihydroxybenzoate (γ-resorcylate; Yoshida et al., 2004 , 2007 ). The polypeptide chain of the enzyme consists of 351 amino-acid residues (Yoshida et al., 2007 ). The amino-acid sequence of the enzyme is homologous to those of maleylacetate reductases from Ralstonia eutropha JMP134 (46%; Seibert et al., 1993 ), Pseudomonas sp. strain B13 (45%; Kasberg et al., 1997 ) and Agrobacterium tumefaciens (90%; Parke, 1995 ). These homologous enzymes are known to be dimers consisting of two identical subunits. It is also known that they are inhibited by thiol-blocking reagents such as p-chloromercuribenzoate and Hg2+ (Gaal & Neujahr, 1980 ; Kaschabek & Reineke, 1993 ; Seibert et al., 1993 ), indicating that the cysteine residue is probably necessary for the catalytic activity of maleylacetate reductase. The sequence homology suggests that these properties might be conserved in the present enzyme GraC. GraC may be a dimeric enzyme. The highly conserved cysteine residue Cys242 is found in the C-terminal part (Yoshida et al., 2007 ). In addition, the sequence motif of the NAD-binding fingerprint, as proposed by Wierenga et al. (1986 ), is found in the region Asp87–Glu123 in the N-terminal part. These findings suggest that the subunit consists of two domains: an N-terminal NAD-binding domain and a C-terminal active-site domain. Here, we report the crystallization and preliminary X-ray diffraction studies of maleylacetate reductase from Rhizobium sp. strain MTP-10005. The X-ray crystal structure of the present enzyme is expected to reveal structural features that may be common to maleylacetate reductases from other aerobic microorganisms.
Expression of the protein was performed as described previously (Yoshida et al., 2007 ). The protein was expressed without an inducer because the cloned gene construct does not require an inducer for expression. Escherichia coli BL21 Star (DE3) cells harbouring a recombinant plasmid pET14b carrying the graC gene were grown in ampicillin-containing LB medium at 310 K. Growth was continued until the optical density reached approximately 0.6 at 600 nm, after which the cultures were placed at 288 K and cultivated for 24 h so as to increase the soluble portion of the protein. After cultivation, the cells were collected by centrifugation (6500g, 15 min, 277 K), suspended in 20 mM Tris–HCl buffer pH 8.0, washed and repelleted. The washed cells were suspended in buffer A (20 mM imidazole, 50 mM NaH2PO4.H2O, 300 mM NaCl pH 8.0) and disrupted with an ultrasonic disintegrator. The cell debris was removed by centrifugation (16 000g, 15 min, 277 K) to obtain the crude enzyme in the supernatant solution.
Purification of the C-terminally His-tagged protein was performed by chromatography at 277 K and a flow rate of 0.5 ml min−1. The crude enzyme was applied onto an Ni–NTA agarose (Qiagen) column equilibrated with buffer A and washed with buffer B (50 mM imidazole, 50 mM NaH2PO4.H2O, 300 mM NaCl pH 8.0). The adsorbed proteins were eluted with buffer C (150 mM imidazole, 50 mM NaH2PO4.H2O, 300 mM NaCl pH 8.0). The eluted protein fractions showing a peak in 280 nm absorbance were combined and concentrated by ultrafiltration. The concentrated protein solution was applied onto a PD-10 desalting column equilibrated with 50 mM Tris–HCl buffer pH 8.0. The GraC protein was eluted with the same buffer. The purity of the protein was determined by 13%(w/v) SDS–PAGE analysis. The protein solution was concentrated to 1 mg ml−1 and stored at 193 K until the crystallization experiments.
Initial crystallization experiments were performed by the hanging-drop vapour-diffusion method using Crystal Screen I (CS I) and Crystal Screen II (CS II) (Hampton Research, California, USA). The protein concentration was adjusted to 10 mg ml−1 (CS I Nos. 1–18 and 25–42) or 5 mg ml−1 (CS I Nos. 19–24 and 43–50, CS II) in 50 mM Tris–HCl buffer pH 8.0. Drops consisting of 1 µl protein solution and 1 µl reservoir solution were equilibrated against 500 µl reservoir solution at 293 K. Small rhombohedron-shaped crystals were obtained after several days with CSI No. 16 and CS II No. 32 and needle crystals appeared within a week with CS I Nos. 6, 9, 15 and 18 and CS II No. 26. The crystallization conditions were optimized based on those of CS II No. 32 in conjunction with an additive screen (Additive Screen, Hampton Research). The final conditions, after optimization, produced rhombohedron-shaped crystals with approximate dimensions of 0.30 × 0.20 × 0.05 mm at 293 K in 3 d using the sitting-drop vapour-diffusion method (Fig. 1 ). Drops of 1 µl protein solution at 8 mg ml−1 (in 50 mM Tris–HCl buffer pH 8.0) and 1 µl reservoir solution were equilibrated against 500 µl reservoir solution consisting of 1.1–1.4 M ammonium sulfate, 0.1 M sodium chloride, 2%(w/v) benzamidine–HCl and 0.1 M Na HEPES pH 7.5. The drops were microseeded using the original crystals grown in CS II No. 32.
Diffraction experiments were performed on beamline NW12A, Photon Factory PF-AR, Tsukuba, Japan. A crystal with typical dimensions of 0.30 × 0.20 × 0.05 mm was soaked in a cryoprotectant solution consisting of 1.4 M ammonium sulfate and 25%(v/v) glycerol for less than 1 min and flash-cooled in a nitrogen stream at 100 K. Diffraction data were collected at a wavelength of 1.000 Å using a Quantum 210 CCD detector (ADSC Inc., California, USA) set at a crystal-to-detector distance of 166.2 mm. A data set was collected using 180 frames with a 1.0° oscillation angle and an exposure time of 0.35 s. All diffraction images were processed to 1.79 Å resolution with HKL-2000 (Otwinowski & Minor, 1997 ).
The crystal belongs to space group C2, with unit-cell parameters a = 56.85, b = 121.13, c = 94.09 Å, β = 101.48°. The statistics of data collection and processing are summarized in Table 1 . Assuming that the crystal contains four dimeric molecules in the unit cell, the V M value is 2.18 Å3 Da−1, which is acceptable for a protein crystal (Matthews, 1968 ). A map of the self-rotation function at 3.0 Å resolution gave a low peak in the κ = 180° plane showing the twofold symmetry relation between the subunits in the molecule. This result may suggest that the structures of the subunits in the dimeric molecule may be slightly different from each other in each domain of the subunit and/or in the relative arrangement between the domains in the subunit. Crystallographic structural studies of GraC from Rhizobium sp. strain MTP-10005 have been initiated by searching for a homologous protein of known structure for molecular replacement and by preparing heavy-atom derivatives for isomorphous replacement.
This work was supported in part by a Grant-in-Aid for the Global COE Program ‘International Center for Integrated Research and Advanced Education in Materials Science’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The diffraction experiments at Photon Factory were approved by the Photon Factory Advisory Committee and the High Energy Accelerator Research Organization (KEK; proposal No. 2006G387).