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The assimilation of aromatic compounds by microbial species requires specialized enzymes to cleave the thermodynamically stable ring. In the recently discovered benzoate-oxidation (box) pathway in Burkholderia xenovorans LB400, this is accomplished by a novel dihydrodiol lyase (BoxCC). Sequence analysis suggests that BoxCC is part of the crotonase superfamily but includes an additional uncharacterized region of approximately 115 residues that is predicted to mediate ring cleavage. Processing of X-ray diffraction data to 1.5 Å resolution revealed that BoxCC crystallized with two molecules in the asymmetric unit of the P212121 space group, with a solvent content of 47% and a Matthews coefficient of 2.32 Å3 Da−1. Selenomethionine BoxCC has been purified and crystals are currently being refined for anomalous dispersion studies.
Benzoate-containing compounds, many of which are derived from decaying plant material, are widely distributed in nature and play a key role in the global carbon cycle. The inherent thermodynamic stability of the aromatic ring restricts nonbiological turnover and consequently the removal of these compounds from the environment relies on microbial metabolic pathways. However, xenobiotic aromatic compounds in the form of industrial effluents, pesticides and solvents are relatively new to the environment and present unique challenges for microbial degradation. A promising strategy to facilitate the degradation of these pollutants is to engineer increasingly efficient metabolic pathways with broadened substrate specificity. As a prerequisite step towards the rational engineering of these biological systems, detailed biochemical and structural descriptions of the enzymes involved are essential.
Microbial degradation of aromatic compounds occurs both aerobically (Bugg, 2003 ; Harwood & Parales, 1996 ; Stanier & Ornston, 1973 ) and anaerobically (Evans, 1977 ; Evans & Fuchs, 1988 ; Harwood & Gibson, 1986 ). However, recent studies have identified a metabolic pathway in Burkholderia xenovorans LB400 (hereafter referred to as LB400) and Azoarcus evansii, termed the benzoate-oxidation (box) pathway, that incorporates features of both (Denef et al., 2005 , 2006 ; Mohamed et al., 2001 ). A unique feature of LB400 is that it contains two paralogous copies of the box pathway: one on the chromosome (box C) and the second on a plasmid (box M). In each case, however, the box pathway catalyzes the aerobic degradation of benzoate to yield succinyl and acetyl coenzyme A (CoA), which are used as metabolic fuels in the Krebs cycle. A salient feature of this pathway is that each intermediate is processed as a CoA thioester, which is predicted to facilitate ring cleavage via a novel dihydrodiol lyase (2,3-dihydro-2,3-dihydroxybenzoyl-CoA lyase/hydratase; BoxC; Gescher et al., 2005 ). Sequence analysis suggests that BoxC is a member of the crotonase superfamily. The closest homologue for which a structure has been solved is the enoyl-CoA hydratase (subunit II) from Geobacillus kaustophilus (PDB code 2ej5), which shows approximately 27% sequence identity over 220 residues. However, BoxC is nearly double the size of a prototypical hydratase (Holden et al., 2001 ) and also includes a central region of approximately 115 residues that has no significant sequence similarity to other members of the crotonase superfamily. Intriguingly, it is this region that is predicted to catalyze the cleavage of the uncommon alicyclic dihydroxylated benzoate ring using Δ3,5,Δ2,4-dienoyl-coenzyme A isomerase-like and 2-ketocyclohexylcarboxyl-CoA hydrolase-like activities (Gescher et al., 2005 ). No structural studies have been reported for a BoxC enzyme from any organism and as a result details of the features that govern catalysis are undefined.
A key step in the assimilation of benzoates by the recently discovered benzoate-oxidation (box) pathway is the cleavage of the dihydroxylated benzoate ring by BoxC. As a first step towards establishing the structural and mechanistic properties of these novel ring-cleaving enzymes, expression, purification and preliminary high-resolution (1.5 Å) X-ray data analysis was carried out on the chromosomally encoded BoxC (BoxCC) from LB400. The preliminary characterization reported here represents a significant step forward in establishing a structural paradigm for aerobic benzoate degradation by the box pathway.
The boxc c (chromosomally encoded boxc) gene was amplified from LB400 genomic DNA and cloned into pET-28a (Novagen) in frame with an N-terminal hexahistidine tag. Sequence analysis confirmed that no mutations were introduced during amplification. Expression of recombinant BoxCC was performed in Escherichia coli BL21 Star (DE3) cells (Invitrogen) grown in 2×YT media (EMD chemicals) supplemented with 50 µg ml−1 kanamycin (Sigma). Transformed cells were initially grown at 310 K to an OD600 of 1, at which point the temperature was lowered to 300 K and BoxCC expression was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) at a final concentration of 0.75 mM. After 12 h, the cells were harvested by centrifugation at 9000g for 15 min and the pellet was resuspended in 20 mM HEPES pH 7.5, 15 mM imidazole, 3 mM β-mercaptoethanol and 500 mM NaCl. The cells were lysed using a French press (SLM Instruments) and the insoluble fraction was removed by centrifugation at 20 000g for 45 min. The supernatant was applied onto Ni–NTA resin (Qiagen) and washed extensively with buffer A (20 mM HEPES pH 7.5, 500 mM NaCl and 3 mM β-mercaptoethanol) supplemented with 20 mM imidazole and BoxCC was eluted with buffer A supplemented with 250 mM imidazole. SDS–PAGE was used to check the purity of BoxCC prior to proteolytic removal of the hexahistidine tag by thrombin. In its mature form following thrombin cleavage, BoxCC includes an additional three amino acids at the N-terminus with Gly and Ser derived from the thrombin site and His contributed from the NdeI site. No additional residues were added at the C-terminus. In the final step, BoxCC was purified on a Superdex S-200 16/60 HiLoad size-exclusion column (GE Healthcare) equilibrated with buffer B (20 mM HEPES pH 7.5, 150 mM NaCl and 3 mM β-mercaptoethanol). Fractions were analyzed by SDS–PAGE, pooled based on purity and concentrated to 13 mg ml−1 in buffer B as determined by A 280.
Expression and purification of selenomethionine BoxCC was carried out as described for native BoxCC with the following changes: the expression vector was transformed into E. coli 834 (DE3) (a methionine auxotroph; Novagen) and grown in SelenoMet medium (AthenaES) supplemented with l-selenomethionine to a final concentration of 40 µg ml−1 (AthenaES).
Initial crystallization trials were set up with commercial screens (Wizard I, II and III from Emerald Biosystems and Index and PEG/Ion Screens from Hampton Research) in 96-well plates (Axygen Biosciences). The final drops consisted of 1.5 µl protein solution (at 13 mg ml−1) and 1.5 µl reservoir solution and were equilibrated against 100 µl reservoir solution. Small crystals of native BoxCC were observed in 23% PEG 3350 with 100 mM Tris pH 8.5 after 3 d at 293 K. Two rounds of streak-seeding were required to sufficiently increase the crystal size (0.2 × 0.1 × 0.2 mm) and quality to enable data collection. For the streak-seeding experiment, approximately a dozen small crystals (0.05 × 0.05 × 0.1 mm) were harvested into 50 µl reservoir solution and crushed with a seed bead (Hampton Research, USA). 0.3 µl of the seed mix was then added to each sitting drop pre-equilibrated with protein and reservoir solution (1:1) with a 3 µl total volume. Optimization of selenomethionine BoxCC crystals is currently under way.
A single native BoxCC crystal (Fig. 2 a) was looped into cryoprotectant consisting of mother liquor supplemented with 20% glycerol for 30 s and flash-cooled directly in the cryostream (100 K). Diffraction data were collected on a Rigaku R-AXIS IV++ area detector coupled to an MM-002 X-ray generator with Osmic ‘blue’ optics and an Oxford Cryostream 700. Each of the 450 images was exposed for 180 s over an oscillation range of 0.5° (Fig. 2 b). Diffraction data to 1.5 Å were processed using CrystalClear/d*TREK (Pflugrath, 1999 ). Data-collection statistics are presented in Table 1 .
The novel ring-cleaving enzyme BoxCC from LB400 was successfully cloned, recombinantly expressed, purified and crystallized. Relative to protein standards, the elution profile of BoxCC from a Superdex 200 size-exclusion column showed the enzyme to be a stable dimer in solution with a molecular weight of approximately 120 kDa (Fig. 1 ), which is consistent with its homologue from A. evansii (Gescher et al., 2005 ). The gel-filtration fractions were analyzed by SDS–PAGE, which showed a predominant band at approximately 60 kDa as expected for the BoxCC monomer (61 579 Da). Streak-seeding produced high-quality crystals that diffracted to 1.5 Å resolution. Processing of the data (Table 1 ) showed that BoxCC crystallized as a dimer in the asymmetric unit of the P212121 unit cell, which was consistent with the observation of a non-origin peak in the self-rotation function, with a solvent content of 47% and a Matthews coefficient of 2.32 Å3 Da−1 (Matthews, 1968 ). Scaling and merging of the data resulted in an overall R merge of 6.2%, with a value of 35.8% in the highest resolution shell (1.55–1.5 Å). Ultimately, structure solution of BoxCC will require experimental phases. To this end, selenomethionine BoxCC protein was purified and crystals are currently being refined for anomalous dispersion synchrotron data collection.
This work was supported by a research grant from the Natural Sciences and Engineering Research Council of Canada to MJB. JB was supported by a University of Victoria Graduate fellowship and MJB is a Canadian Institutes of Health Research New Investigator and a Michael Smith Foundation for Health Research Scholar.