Lactones are a class of structurally diverse molecules that serve essential roles in biological processes ranging from quorum sensing to the aerobic catabolism of aromatic compounds. Not surprisingly, enzymes involved in the bioprocessing of lactones are often targeted for protein engineering studies with the potential, for example, of optimized bioremediation of aromatic pollutants. The enol-lactone hydrolase (ELH) represents one such class of targeted enzymes and catalyzes the conversion of 3-oxoadipate-enol-lactone into the linear β-ketoadipate. To define the structural details that govern ELH catalysis and assess the impact of divergent features predicted by sequence analysis, we report the first structural characterization of an ELH (PcaD) from Burkholderia xenovorans LB400 in complex with the product analog levulinic acid. The overall dimeric structure of PcaD reveals an α-helical cap domain positioned atop a core α/β-hydrolase domain. Despite the localization of the conserved catalytic triad to the core domain, levulinic acid is bound largely within the region of the active site defined by the cap domain, suggesting a key role for this divergent substructure in mediating product release. Furthermore, the architecture of the cap domain results in an unusually deep active-site pocket with topological features to restrict binding to small or kinked substrates. The evolutionary basis for this substrate selectivity is discussed with respect to the homologous dienelactone hydrolase. Overall, the PcaD costructure provides a detailed insight into the intimate role of the cap domain in influencing all aspects of substrate binding, turnover, and product release.
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