Structural and Biophysical Characterization of BoxC from Burkholderia xenovorans LB400: A NOVEL RING-CLEAVING ENZYME IN THE CROTONASE SUPERFAMILY*

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 microorganisms. 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 (2–4). The anaerobic pathway, however, mediates a reductive dearomatization followed by a hydrolytic ring cleavage, as observed in the classical benzoate pathway (5–7). 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 (11–13). 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.