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Structural and Biophysical Characterization of BoxC from Burkholderia xenovorans LB400

The mineralization of aromatic compounds by microorganisms relies on a structurally and functionally diverse group of ring-cleaving enzymes. The recently discovered benzoate oxidation pathway in Burkholderia xenovorans LB400 encodes a novel such ring-cleaving enzyme, termed BoxC, that catalyzes the conversion of 2,3-dihydro-2,3-dihydroxybenzoyl-CoA to 3,4-dehydroadipyl-CoA without the requirement for molecular oxygen. Sequence analysis indicates that BoxC is a highly divergent member of the crotonase superfamily and nearly double the size of the average superfamily member. The structure of BoxC determined to 1.5 Å resolution reveals an intriguing structural demarcation. A highly divergent region in the C terminus probably serves as a structural scaffold for the conserved N terminus that encompasses the active site and, in conjunction with a conserved C-terminal helix, mediates dimer formation. Isothermal titration calorimetry and molecular docking simulations contribute to a detailed view of the active site, resulting in a compelling mechanistic model where a pair of conserved glutamate residues (Glu146 and Glu168) work in tandem to deprotonate the dihydroxylated ring substrate, leading to cleavage. A final deformylation step incorporating a water molecule and Cys111 as a general base completes the formation of 3,4-dehydroadipyl-CoA product. Overall, this study establishes the basis for BoxC as one of the most divergent members of the crotonase superfamily and provides the first structural insight into the mechanism of this novel class of ring-cleaving enzymes.

Aromatic compounds comprise approximately one-quarter of the earth's biomass (1) and are the second most abundant natural product next to carbohydrates. The majority of aromatic compounds in the environment are in the form of the organic polymer lignin that plays a structural role in cross-linking cell wall polysaccharides in plants. Despite the inherent thermostability of the aromatic ring, these naturally occurring compounds are efficiently mineralized by various microorga-nisms. Human-made aromatic compounds, such as those used in industrial processes, however, are often recalcitrant to microbial degradation due to their chemical complexity, decreased bioavailability, and increased thermostability. Moreover, bacteria have only been exposed to these compounds for a relatively short period of time. As a result, these compounds persist in the environment, where they can increase to toxic levels and cause irreversible damage to the biosphere.
The common structural blueprint shared by natural and human-made aromatic compounds is the resonance-stabilized planar ring system. Microorganisms overcome the stability of these aromatic structures by employing specific ring-cleaving enzymes that form part of complex catabolic pathways. Until recently, two general classes of microbial processes were characterized that catalyze the degradation of aromatic compounds. These classifications, termed the aerobic and anaerobic pathways, were based primarily on the mode of initial activation and subsequent cleavage of the aromatic ring. The aerobic pathway, exemplified by the peripheral biphenyl and the central ben-cat pathway, relies on the extensive use of molecular oxygen for both the hydroxylation (activation) and cleavage of the aromatic ring (2)(3)(4). The anaerobic pathway, however, mediates a reductive dearomatization followed by a hydrolytic ring cleavage, as observed in the classical benzoate pathway (5)(6)(7). In both cases, the underlying mechanism incorporates an activation step that renders the ring susceptible to cleavage.
Recently, a third aromatic degradation pathway was identified in Burkholderia xenovorans strain LB400 (LB400) (8 -10) and Azoarcus evansii (11)(12)(13). This novel pathway, termed the box (benzoate oxidation) pathway, incorporates features of both the aerobic and anaerobic pathways, resulting in a hybrid pathway. Microarray analysis of the 9.7-Mb genome of LB400 revealed two paralogous copies of the box pathway, one encoded on chromosome 1 (box c ) and the second on the megaplasmid (box m ) (9). Knock-out studies confirm that both box pathways are capable of assimilating benzoate (10) yet are differentially regulated based on available carbon source and growth phase of the organism (9). Recent structural and biochemical characterization of benzoate CoA ligase (14) and aldeheyde dehydrogenase (15) from the box pathway in LB400 have provided valuable insight into the basis of substrate specificity and details describing the molecular mechanisms.
A unique feature of the hybrid box pathway is the incorporation of both CoA ligation and hydroxylation prior to ring cleavage (16), suggesting that both strategies are important for ring activation. It is noteworthy that although CoA ligation is common in the activation of aromatic acids under anaerobic conditions, it has thus far been unseen in the aerobic degradation of aromatic compounds. Furthermore, investigation of the box pathway intermediates from the related A. evansii demonstrated that the thioesterified dihydrodiol intermediate was not oxidized and rearomatized as normally occurs in aerobic aromatic metabolism (11). Instead, it was shown to be directly cleaved without the requirement of molecular oxygen in a reaction that resulted in the loss of one unit of carbon and oxygen as formate (11). This critical ring cleavage step in the box pathway is catalyzed by BoxC (2,3-dihydro-2,3-dihydroxybenzoyl-CoA lyase/hydrolase) (11), which differs from traditional aerobic and anaerobic ring-cleaving enzymes in that oxygen is not used in catalysis, and the ring substrate is only partially reduced. Based on sequence analysis, BoxC is assigned to the crotonase superfamily. The cleavage reaction catalyzed by BoxC, however, suggests that BoxC defines a new mechanistic niche and intriguingly is one of the four outstanding crotonase superfamily members for which no structural information exists (17).
A mechanism for BoxC from A. evansii was recently proposed based on the identification of chemical species using NMR and mass spectrometry (11). In the absence of structural information of BoxC, however, the mechanistic details, including the identity of the catalytic residues, remain undefined. To investigate the detailed molecular mechanism of BoxC, we carried out a structural and biophysical analysis complemented with molecular docking. The resulting data provide a compelling mechanistic model with the identification of key catalytic residues and active site structure that stabilize proposed transition state intermediates. Furthermore, the 1.5 Å resolution structure of BoxC reveals intriguing divergent architectural features with respect to other members of the crotonase superfamily. Overall, this study provides the first structural characterization of the novel BoxC family of enzymes and is interpreted with respect to the proposed molecular mechanism and divergence within the crotonase superfamily.

EXPERIMENTAL PROCEDURES
Protein Production, Purification, and Crystallization-The chromosomally encoded boxc c gene from B. xenovorans LB400 was cloned into pET-28a(ϩ) (Novagen) in frame with an N-terminal hexahistidine tag. Sequence analysis confirmed that no mutations were introduced during cloning. Native (18) and selenomethionine BoxC C were produced and purified using nickel affinity and size exclusion chromatography. Crystals of native BoxC C were obtained using the vapor diffusion method in 25% polyethylene glycol 3350 and 100 mM Tris, pH 8.5 (18). Crystals of selenomethionine BoxC C were initially obtained by seeding with native BoxC C crystals, followed by two additional rounds of microseeding to obtain diffraction quality crystals.
Data Collection, Structure Solution, and Refinement-Diffraction data for native BoxC C crystals were collected as described previously (18). Diffraction data for selenomethionine BoxC C were collected on beamline X8C at the National Synchrotron Light Source (Brookhaven National Laboratories) at the optimized wavelength of 0.9794 Å for the fЉ selenium edge. Data processing was carried out using Crystal Clear/ d*trek (19). A total of 12 selenium sites (six from each monomer) were identified and refined using autoSHARP (20), resulting in a figure of merit of 0.297. High quality phases were obtained following density modification and 2-fold NCS averaging that enabled building and registering of ϳ70% of the backbone using ARP/Warp (21). The remaining structure was built manually, and solvent atoms were selected using COOT (22) and refined with REFMAC (23) to an R cryst of 18.7% and an R free of 20.9%. In total, 174,578 reflections were used in refinement selected with a cut-off of 2.0. All solvent atoms were inspected manually before deposition. Stereochemical analysis of the refined BoxC C structure was performed with PRO-CHECK (24) and SFCHECK in CCP4 (25), with the Ramachandran plot showing excellent stereochemistry with 99.8% of the residues in the most favored and additional allowed conformations and no residues modeled in disallowed orientations. Overall, 5% of the reflections were set aside for calculation of R free . Data collection statistics are presented in Table 1.
Isothermal Titration Calorimetry (ITC) 3 -ITC was performed using a VP TM isothermal titration calorimeter (Micro-Cal, Northampton, MA). All samples were characterized in 20 mM Tris buffer, pH 8.5, supplemented with 150 mM NaCl. Protein and ligand solutions were filtered and degassed immediately prior to use. Titrations were performed by injecting 20-l aliquots of ligand solution into the ITC sample cell containing 200 M BoxC C at 22°C. All ITC data were corrected for the heat of dilution of the titrant by subtracting the heats generated by titrating the ligand into buffer alone. The equilibrium association constant and the stoichiometry were determined by curve fitting. Two independent titration experiments were performed per ligand, and the average was taken. Thermodynamic parameters were calculated from the Gibbs free energy equation, ⌬G ϭ ϪRT ln K a ϭ ⌬H Ϫ T⌬S.
Bioinformatics and Molecular Modeling-Multiple sequence alignments and the associated neighbor joining trees were determined by ClustalW (26), using the method of Saitou and Nei (27). Buried surface area for the BoxC C dimer interface was calculated using the Protein-Protein interaction analysis server (available on the World Wide Web). Docking was performed with the program Molegro virtual docker (28), using the molecular docking algorithm Moldock score. Initially, water, glycerol, and ␤-mercaptoethanol (␤-Me) molecules were removed from the structure coordinates of BoxC C . Prior to docking, the structure of the 2,3-dihydro-2,3-dihydroxybenzoyl-CoA was built and energy-minimized at the MP2/6-31G* level using Gaussian 03 (29). Ligand binding cavities were identified using the Molegro Van der Waals molecular surface prediction algorithm with a grid resolution of 0.5 Å. A total of 50 docking runs with a population size of 200 were calculated over a 12-Å radius surrounding the predicted active site cavity with a grid resolution of 0.2 Å and a maximum of 10,000 iterations per position. Similar positions were clustered using a root mean square deviation of 1.5 Å. Prepositioned ligands were randomized in the predicted active site cavity prior to each docking run, and docking was constrained to the predicted active site cavity. In order to verify that positions resulting from in silico docking represent correctly bound conformations, each position was visually inspected and compared. Positions were also inspected and compared with the rerank score algorithm, protein interaction, hydrogen bonding, and affinity interaction energies and ordered by the energy of interaction protein-ligand. Complexes were optimized by using Moloc software (30,31) with standard force field and optimization parameters. During energy minimization, the positions of amino acid side chains were fixed while allowing all ligand atoms to move.

Overall Structure
BoxC C crystallized as a dimer in the asymmetric unit of the primitive orthorhombic (P2 1 2 1 2 1 ) cell. The structure was solved by single anomalous wavelength dispersion, using selenomethionine-derivatized BoxC C . The final model starts at Pro 10 (monomer A) and Ala 9 (monomer B) and extends through Val 556 (Fig. 1A, left). Included in the final model are six molecules of glycerol and four molecules of ␤-Me. It is noteworthy that in each monomer a ␤-Me molecule is coordinated to the side chains of Cys 90 and Cys 111 (Fig. 1A, right). No ␤-Me is coordinated to the remaining four cysteines, suggesting that Cys 90 and Cys 111 are particularly reactive.
The formation of the BoxC C dimer results in an extensive buried surface area of ϳ3900 Å 2 , consistent with our observation that BoxC C elutes as a stable dimer from a size exclusion column (18). The dimeric interface of BoxC C is formed by a network of interlocking ␣-helices, with ␣6, ␣7, ␣19, and ␣20 contributing the majority of the buried surface area (Fig. 1B,  left). Clear electron density is observed for each interface residue, including large polar residues, such as Arg 191 and His 192 , that bridge the two monomers through an extensive solvent network that appears to increase shape complementarity of the interface (Fig. 1B, right). Nearly 62% of the residues responsible for dimer formation are conserved in BoxC orthologs, suggesting that the dimeric structure of this novel group of ring-cleaving enzymes will be consistent. In the broader context of the crotonase superfamily, the dimeric form is rare, with most members adopting trimer, tetramer, or hexamer (dimers of trimers) forms (32)(33)(34)(35)(36)(37)(38)(39)(40) and, in one case, proposed to form a trimer of dimers (41). Only the carboxyltransferase subunits for the acetyl-CoA carboxylase from Saccharomyces cerevisiae (42) and the ␣-subunit of glutaconyl-CoA decarboxylase (GCD␣) from A. fermentans (43), which are subunits of larger multifunctional enzymes, are reported to be dimers. The 2-fold symmetry of the BoxC C dimer is mirrored in each of the monomeric subunits. The N-terminal ( Fig. 1C; residues 1-250) and C-terminal ( Fig. 1C; residues 251-507) domains are related by 180°rotations about the vertical and horizontal axes. A central helical bundle incorporating helix ␣20 formed by the terminal 49 residues of the C terminus ( Fig. 1C; magenta) comprises the intramolecular interface. The N and C termini share only 18% sequence identity but adopt a conserved ␣/␤ architecture with a root mean square deviation of 1.69 Å over 131 C␣ atoms (Fig. 1C, right). The N-terminal domain consists of a seven-stranded twisted ␤-sheet sandwiched between ␣-helices and an additional two-stranded ␤-sheet positioned perpendicular to the main ␤-sheet. A structural comparison indicates that the ␤ substructures surrounded by ␣-helices are conserved in the crotonase superfamily (44). Interestingly, the seven-strand twisted ␤-sheet in the C-terminal domain differs from the N-terminal domain in that it is longer and incorporates an additional antiparallel ␤-strand while lacking the second perpendicular ␤-sheet. In addition, a short helix in the N-terminal domain is substituted for a more extended helix in the C-terminal domain, resulting in a reorganized topology and surface structure.

Structural Homology
Divergence among BoxC Orthologs-To evaluate the conserved architectural features of BoxC C in the context of its closely related orthologs, all of which exhibit greater than 57% identity, we mapped the sequence alignment results onto the Connolly surface (45) calculated for BoxC C (Fig. 2). A striking demarcation is observed between regions of high (50 -61%; magenta) and low (15-20%; gray) sequence identity. It is clear from this analysis that maximum sequence divergence is localized to the C-terminal domain with the exception of helix ␣18 (Fig. 2, arrow), which is composed of conserved acidic residues (Asp 477 , Asp 480 , Glu 481 , Glu 487 , and Glu 488 ). In the context of the BoxC C dimer, this negatively charged helix is solvent-exposed, suggesting a potentially important functional role. The majority of the conserved residues map to the dimer interface, contributing 81% of the overall buried surface area. The majority of the divergent region, however, is distal to the dimer interface. We hypothesize that this divergent region serves an ancillary role by providing a structural scaffold for the N-terminal domain and the dimerization interface. In this regard, it could thus also contribute indirectly in forming a docking surface for mediating a higher order complex with BoxA/B, as suggested previously (46). A homologous docking site on the dimeric structure has also been hypothesized for the GCD␣ subunit (43).
A Unique Member within the Crotonase Superfamily-Members of the crotonase superfamily exhibit as little as 20% sequence identity but incorporate conserved structural hallmarks. To determine the evolutionary relationship between BoxC C and each mechanistic class of the crotonase superfamily (17), we generated phylogenetic trees using the entire sequence of BoxC C (residues 1-556) and the individual N-and C-terminal domains (Fig. 3). BoxC C is approximately double the size of most members of the crotonase superfamily with the exception of the dioxygenase (47) and the carboxyltransferase subunit of biotin-dependent carboxylases (42,43,48). The carboxyltransferase subunits, however, have been omitted from the alignment, since they represent subunits of larger multifunctional enzymes. The N-terminal region (residues ϳ50 -250) of BoxC C shows highest similarity with the enoyl-CoA hydratases/ isomerases, whereas the C-terminal region (residues ϳ360 -475) shares homology with 1,4-dihydroxynaphthoyl-CoA synthase and an enoyl-CoA hydratase. Residues 260 -350 show no significant identity to any particular class, suggesting that this region is structurally and/or functionally specific to BoxC C . It is interesting to note, however, that this region lies in the most divergent stretch within the BoxC C orthologs (Fig. 2), consistent with a role as a support scaffold. A similar scenario was recently observed in dihydroxyphenylglyoxylate synthase, where the initial one-third of the sequence (N-terminal) shares no sequence homology to any known proteins (47). With the active site localized in the C termini of dihydroxyphenylglyoxylate synthase, this novel region has not been ascribed any specific role. Overall, the phylogram clearly demonstrates that of the proteins analyzed, BoxC C is the most divergent member of the crotonase superfamily (Fig. 3A). Furthermore, the C-terminal domain of BoxC C is significantly more divergent than the N-terminal domain (Figs. 3, B and C), displaying a similar pattern to that observed for the BoxC C orthologs (Fig. 2).

Identifying and Mapping the Active Site
A Conserved Structural Scaffold-In the absence of the BoxC C co-structure, we used structural overlays with members of the crotonase superfamily for which active sites have been structurally characterized to define the location of the active site (Fig. 4, A-H, black arrows). Despite the mechanistically divergent nature of these enzymes, it is clear that members of the crotonase superfamily share a common catalytic scaffold (49). Based on these overlays, the active site in BoxC C maps to the N-terminal domain and is completely encompassed by a single monomer. The strict localization of the active site to a single monomer is rare in the crotonase superfamily (33,50), with most members presenting an active site that spans a multimeric interface (34,35,38,40,(51)(52)(53). The calculated volume of the predicted active site is ϳ300 Å 3 and forms an 18-Å-deep tunnel, consistent with the ability to coordinate the extended 2,3-dihydro-2,3-dihydroxybenzoyl-CoA/benzoyl-CoA dihydrodiol substrate. An open pocket positioned at the bottom of the active site tunnel appears sufficiently large to accommodate the linear aliphatic chain that results from cleavage of the dihydroxylated ring.
To validate the location and structure of the active site and identify potential catalytic residues, we used a molecular docking approach with the native substrate. Previous NMR studies by Gescher et al. (11) indicated that the dihydrodiol of the native BoxC C substrate adopts the cis conformation. We therefore used the cis-isomer of the dihydrodiol, including both of the possible diastereoisomers, 2S,3R and 2R,3S, in our docking scenarios. Energy minimizations confirmed that our predicted active site location resulted in the highest score with no steric clashes. A detailed analysis indicates that the lower portion of the active site (Fig. 4I) is completely hydrophobic and defined by residues Ile 96 , Leu 99 (helix ␣2), Phe 110 (␣3), Leu 172 (␣5), Leu 174 (loop region connecting ␣5 and ␣6), and Phe 528 (␣20). This structural configuration provides a rationale for why BoxC C is selective for the cis-isomer 2R,3S, such that neither hydroxyl is directed toward the hydrophobic region. In our model, helix ␣4 contributes structural integrity to the upper portion of the substrate binding tunnel and encodes one residue (Gly 143 ) for the putative oxyanion hole. It is noteworthy that the structurally homologous helix ␣4 is referred to as the "active site helix" (35,38), where the dipole and hydrogen bonding interactions are implicated in polarizing the thioester carbonyl of the aromatic ring substrate in 4-chlorobenzoyl-CoA dehalogenase (34).
A similar binding mode for CoA is observed in members of the crotonase superfamily. The specific residues, however, are unique to the individual homologues. In our docked model, CoA adopts a hook shape binding mode with the pantothenic acid moiety much less solvent-exposed than observed with most members of the crotonase superfamily. The key residues in BoxC C that appear to be responsible for coordinating the CoA are Lys 51 , Ser 92 , and Ser 165 , each of which is highly conserved within the BoxC orthologs. Stabilizing hydrogen bonds are formed between the backbone carbonyl of Lys 51 and ␥O of Ser 92 and the pantothenate moiety and between the ␥O of Ser 165 and the adenine ring.
Conserved Catalytic Residues-To define catalytically important residues in the active site of BoxC C , we compared our docked model with the homologous enoyl-CoA hydratase (ECH) (35) and dienoyl-CoA isomerase (DCI) (38) (Fig. 4I). ECH is a classical member of the crotonase superfamily, and sequence comparisons indicate conservation of key catalytic residues, whereas DCI catalyzes a similar reaction to that proposed as the initial reaction in BoxC C . Interestingly, a similar arrangement of hydrophobic to hydrophilic residues is observed in ECH and DCI, where the pockets are largely hydrophobic with strategically positioned polar catalytic residues to catalyze stereo-specific reactions (35,38). The critical conserved elements in the active site of BoxC C , however, are a pair of acidic residues that participate in acidbased catalysis in related crotonase superfamily members (35,38). Glu 146 in BoxC C adopts a conserved spatial orientation with Glu 144 in ECH and Asp 176 in DCI, whereas Glu 168 in BoxC C superimposes with Glu 164 in ECH and Glu 191 in DCI (Fig. 4I). The proximity of Cys 111 to the modeled substrate provides a powerful nucleophile that that may be involved in catalysis and also rationalizes why we were unable to obtain a co-crystal structure with BoxC C . As shown in Fig. 1A, Cys 111 is covalently modified by the  Fig. 1. A, phylogram generated using the entire length of BoxC C . B, phylogram generated using residues 1-250 of BoxC C . C, phylogram generated using residues 251-507. The members of the crotonase superfamily included in the phylogenetic analysis are 4-chlorobenzoyl-CoA dehalogenase (CBD) (UniProtKB/TrEMBL A5JTM5); methylmalonyl-CoA decarboxylase (MMCD) (UniProtKB/TrEMBL B5YQB2); ECH (UniProtKB/Swiss-Prot P14604); DCI (UniProtKB/Swiss-Prot Q62651); dihydroxyphenylglyoxylate synthase (DpgC) (UniProtKB/TrEMBL Q8KLK7); hydroxylcinnamoyl-CoA hydrataselyase (HCHL) (UniProtKB/TrEMBL O69762); carboxymethylproline synthase (CarB) (UniProtKB/TrEMBL Q9XB60); 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB) (UniProtKB/TrEMBL Q2FI32); 6-oxo-camphor hydrolase (OCH) (UniProtKB/TrEMBL Q93TU6), and 2-ketocyclohexanecarboxyl-CoA hydrolase (BadI) (UniProtKB/TrEMBL O07456). Overall, BoxC C is the most divergent member of the crotonase superfamily, with the C-terminal domain being more highly divergent than the N-terminal domain.
required crystallization additive, ␤-Me, thereby preventing proper coordination with the substrate analog.

Ligand Binding Studies
ITC-Both the substrate analog benzoyl-CoA and CoA alone were used to complement the structural studies and validate the molecular docking solution. We were unable to use the native substrate (2,3-dihydro-2,3-dihydroxybenzoyl-CoA/benzoyl-CoA dihydrodiol), since it is commercially unavailable, and the chemical synthesis requires a complex biotransformation step to produce the cis form of the dihydrodiol.
Benzoyl-CoA bound to BoxC C with a K d of 116.4 M Ϯ7 M (Fig. 5). The stoichiometry was determined to be 0.90 Ϯ 0.0, consistent with the presence of one active site per monomer as predicted from our structural overlays. In the only other study of a BoxC family member, the K m for the enzymatically synthe-sized native substrate was determined to be 17 Ϯ 2 M (11). Overall, the binding of benzoyl-CoA to BoxC C is enthalpically driven with an accompanying favorable change in entropy. The combination of a relatively small enthalpy change with a favorable entropic contribution suggests that the hydrophobic tail of benzoyl-CoA is the primary source of interactions with BoxC C , consistent with our molecular docking solution. It should be noted that the thermodynamic profile of the native substrate, which incorporates a dihydroxylated benzoyl ring (Fig. 5B,  inset), will display an increased enthalpy due to the additional hydroxyl groups available for coordination and will bind with a lower K d . We also determined that CoA by itself interacts with BoxC C with nearly 25-fold lower affinity (ϳ3 mM; data not shown). These results suggest a limited role for CoA in substrate recognition and are consistent with previous studies of a BoxC C ortholog, where acetoacetyl-CoA, which is a potential inhibitor of ECH, did not inhibit enzyme activity (11). Additionally, the crotonase superfamily member 2-ketocyclohexanecarboxyl-CoA hydrolase was shown to be unreactive with free 2-ketocyclohexanecarboxyl-CoA hydrolase, acetoacetyl-CoA, cyclohex-1-enecarboxyl-CoA, and 2-hydroxycyclohexane carboxyl-CoA (54), indicating that, although CoA is important for catalysis, it plays only a minor role in mediating enzyme substrate binding.

Proposed Catalytic Mechanism
It has been proposed that BoxC catalyzes both an isomerization (38) and hydrolytic deformylation (54) reaction in converting the dihydroxylated ring into the linear aliphatic chain (11). Intriguingly, both reactions are catalyzed by members of the crotonase superfamily and are CoA-dependent.
By incorporating NMR studies carried out by Fuchs and co-workers (11) with our high resolution crystal structure and modeling data, we propose a catalytic mechanism with putative functions ascribed to specific active site residues (Fig. 6). In the initial catalytic step of BoxC C , the dihydroxylated ring of the substrate is deprotonated at either the C-2 or C-3 position. Hydrogen bonding interactions involving these hydroxyl groups will play an important role in biasing the initial attack. We propose that the first step involves deprotonation of the hydroxyl at C-2 by the conserved Glu 146 (Fig. 6, step I). A nearby arginine, Arg 118 , probably facilitates this step by lowering the pK a of Glu 146 to yield a stronger base. Alternatively, it is conceivable that deprotonation is initiated at the C-3 hydroxyl by Cys 111 . In this scenario, Cys 111 probably proceeds through an activated water that would be required to bridge the ϳ3.5-Å distance between the thiol group and the C-3 hydroxyl of the substrate. By comparison, the OE1 group of Glu 146 is positioned ϳ2.7 Å from the C-2 hydroxyl, enabling direct deprotonation of the substrate. The initial deprotonation by Glu 146 results in an oxyanion intermediate on the carbonyl oxygen of the thioester bond that is stabilized by an oxyanion hole formed by the backbone amino groups of Ala 94 and Gly 143 . It is noteworthy that both of these residues share structural equivalents with many members of the superfamily where the oxyanion hole is a required mechanistic feature (17).
In our model, the deprotonation at the C-2 hydroxyl is followed by a second deprotonation at the C-3 hydroxyl (step II). Although it is conceivable that step II is catalyzed by the second conserved glutamate, Glu 168 , we propose that a proton shuttle between the pair of glutamates (Glu 146 and Glu 168 ) resets Glu 146 , allowing it to act as a base in the second deprotonation event. Following each deprotonation event, Glu 168 delivers the proton back to the substrate to enable restructuring of the double bonds and resetting the glutamate proton shuttle. In the event the initial deprotonation is mediated by Cys 111 at the C-3 hydroxyl, the second deprotonation at the C-2 hydroxyl would probably be carried out by Glu 146 .
Following the deprotonation events in steps I and II, the resulting aldehyde groups of Compound A will exist in equilibrium with water, as shown in step III. For the sake of clarity, we have only shown one of the equilibrium products (Compound B), which ultimately undergoes a third and final deprotonation, resulting in release of the formyl group (HCOOH) (step IV). The scenario depicted in Fig. 6 involves Cys 111 as mediating the final deprotonation event. As discussed previously, the distance of Cys 111 to the modeled substrate suggests that it functions through a catalytic water, which, in the activated state, abstracts the proton from one of the C-2 hydroxyls. The flexible side chain of a nearby lysine (Lys 107 ) may lower the pK a of Cys 111 , facilitating its role as a general base. The incorporation of an activated water molecule in our proposed catalytic mechanism is consistent with the chemical requirement to yield formic acid, as suggested previously (11). If the initial deprotonation occurs at the C-3 via Cys 111 , then this third deprotonation step is likely to be mediated by Glu 168 . In a similar fashion to step I, the oxyanion intermediate formed in step IV will be stabilized by the oxyanion hole. Upon the addition of a proton, Compound C decomposes (step V) to Compound D (3,4-dehydroadipyl-CoA), as indicated from previous NMR experiments (11).
The pair of conserved glutamates (Glu 146 and Glu 168 ) along with Cys 111 probably represent the key catalytic residues in the active site of BoxC C . The incorporation of an active site cysteine in the crotonase superfamily is rare, since it has only been described for carboxymethylproline synthase, although recent studies have assigned the catalytic role to a histidine (52).
BoxC C also offers an array of polar residues, including Tyr 55 , Thr 114 , Thr 178 , and Gln 524 , that may prove essential in stabilizing and coordinating reaction intermediates. Sitedirected mutagenesis combined with biochemical characterization will ultimately be required to explicitly define the roles of the individual active site residues.

Conclusion
The 1.5 Å resolution crystal structure of the novel ring-cleaving enzyme BoxC C reveals an intriguing structural divergence and establishes it as a unique member of the crotonase superfamily. By complementing the high resolution structural data with ITC and molecular docking, we are able to propose catalytic roles for specific active site residues. These data extend the initial work of Fuchs and co-workers (11) in proposing a detailed molecular mechanism for BoxC and of Schofield and co-workers (17) in identifying BoxC as a promising target for structural and mechanistic elucidation in the broader context of the crotonase superfamily.