Abstract
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 A 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.
Highlights
Aromatic compounds comprise approximately one-quarter of the earth’s biomass [1] and are the second most abundant natural product next to carbohydrates
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 third aromatic degradation pathway was identified in Burkholderia xenovorans strain LB400 (LB400) (8 –10) and Azoarcus evansii [11,12,13]
Summary
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. 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. 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
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