Aromatic compounds comprise approximately one quarter of the Earth's biomass and thus play a critical role in the biogeochemical carbon cycle. These compounds are degraded almost exclusively by specialized microbial enzymes that are part of complex metabolic pathways. Detailed characterization of these enzymes is both a gateway to understanding a biological process fundamental to nature and a platform for bioengineering applications in bioremediation. Recently, a novel pathway was shown to metabolize two key aromatic intermediates: Benzoate and Benzoyl-Coenzyme A. Designated as the box pathway (benzoate oxidation), this metabolic conduit incorporates in succession; CoA-ligation, oxygenation, ring cleavage and neutralization of the aldehydic ring cleavage product, catalyzed by a Benzoate Coenzyme A Ligase (BCL), BoxAB, BoxC and an Aldehyde Dehydrogenase (ALDH) respectively. Collectively, these steps define the initial and unique segment of the box pathway. The objective of the research described here was to establish a molecular blueprint of the substrate binding pocket of the initial BCL and elucidate mechanistic details for both BoxC and ALDH enzymes from Burkholderia xenovorans LB400 through in-depth structural and functional characterizations. An intriguing feature of the box pathway in LB400 is a paralogous genetic organization. Functional studies on the BCL paralogs (BCLM and BCLC) show that BCLM is more active towards benzoate than BCLC. Structural analysis of the 1.84 Å resolution co- crystal structure of BCLM with benzoate reveals that the substrate binding pocket is closely contoured to bind benzoate, leaving little room to accommodate substituted benzoates, especially in the para position owing to a histidine (H339) residue that renders the pocket particularly shallow. Overall, while corroborative, the structural data provides a molecular rationale to our functional data where both the BCLs were seen to be highly specific for benzoate. Structural analysis of the 1.5 Å resolution crystal structure of the novel ring cleaving BoxC reveals an intriguing structural demarcation consistent with the primary sequence based divergence of BoxC within the crotonase superfamily. A highly divergent region in the C-terminus likely serves as a structural scaffold for the conserved N-terminus that harbors the active site. Isothermal titration calorimetry and molecular docking simulations contribute to a detailed view of the active site resulting in a compelling mechanistic model involving a pair of conserved glutamates (E146 and E168) and a novel cysteine (C111). Lastly, the 1.6 Å resolution co-crystal structure of ALDHC with NADPH and PEG allows identification of residues that are involved in rendering ALDHC selective for NADP+ and linear, medium to long chain aldehydes, as observed in our initial kinetic analyses. Functional and structural characterization of strategic ALDHC mutants enables us to propose a detailed reaction mechanism which involves the essential roles for C296 as the nucleophile, E257 as the general base and a proton relay network anchored by E496 and supported by E167 and K168. Overall, this research provides a molecular blueprint for three key box enzymes, thereby enhancing our understanding of central aromatic metabolism. / Graduate