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Abstract: The recently identified benzoate oxidation (box) pathway in Burkholderia xenovorans LB400 (LB400 hereinafter) assimilates benzoate through a unique mechanism where each intermediate is processed as a coenzyme A (CoA) thioester. A key step in this process is the conversion of 3,4-dehydroadipyl-CoA semialdehyde into its corresponding CoA acid by a novel aldehyde dehydrogenase (ALDH) (EC 1.2.1.x). The goal of this study is to characterize the biochemical and structural properties of the chromosomally encoded form of this new class of ALDHs from LB400 (ALDHC) in order to better understand its role in benzoate degradation. To this end, we carried out kinetic studies with six structurally diverse aldehydes and nicotinamide adenine dinucleotide (phosphate) (NAD + and NADP +). Our data definitively show that ALDHC is more active in the presence of NADP + and selective for linear medium-chain to long-chain aldehydes. To elucidate the structural basis for these biochemical observations, we solved the 1.6-Å crystal structure of ALDHC in complex with NADPH bound in the cofactor-binding pocket and an ordered fragment of a polyethylene glycol molecule bound in the substrate tunnel. These data show that cofactor selectivity is governed by a complex network of hydrogen bonds between the oxygen atoms of the 2′-phosphoryl moiety of NADP + and a threonine/lysine pair on ALDHC. The catalytic preference of ALDHC for linear longer-chain substrates is mediated by a deep narrow configuration of the substrate tunnel. Comparative analysis reveals that reorientation of an extended loop (Asn478-Pro490) in ALDHC induces the constricted structure of the substrate tunnel, with the side chain of Asn478 imposing steric restrictions on branched-chain and aromatic aldehydes. Furthermore, a key glycine (Gly104) positioned at the mouth of the tunnel allows for maximum tunnel depth required to bind medium-chain to long-chain aldehydes. This study provides the first integrated biochemical and structural characterization of a box-pathway-encoded ALDH from any organism and offers insight into the catalytic role of ALDHC in benzoate degradation. [Copyright &y& Elsevier]

Abstract: Xenobiotic aromatic compounds represent one of the most significant classes of environmental pollutants. A novel benzoate oxidation (box) pathway has been identified recently in Burkholderia xenovorans LB400 (referred to simply as LB400) that is capable of assimilating benzoate and intimately tied to the degradation of polychlorinated biphenyls (PCBs). The box pathway in LB400 is present in two paralogous copies (box M and box C) and encodes eight enzymes with the first committed step catalyzed by benzoate CoA ligase (BCL). As a first step towards delineating the biochemical role of the box pathway in LB400, we have carried out functional studies of the paralogous BCL enzymes (BCLM and BCLC) with 20 different putative substrates. We have established a structural rationale for the observed substrate specificities on the basis of a 1.84 Å crystal structure of BCLM in complex with benzoate. These data show that, while BCLM and BCLC display similar overall substrate specificities, BCLM is significantly more active towards benzoate and 2-aminobenzoate with tighter binding (K m) and a faster reaction rate (V max). Despite these clear functional differences, the residues that define the substrate-binding site in BCLM are completely conserved in BCLC, suggesting that second shell residues may play a significant role in substrate recognition and catalysis. Furthermore, comparison of the active site of BCLM with the recently solved structures of 4-chlorobenzoate CoA ligase and 2, 3-dihydroxybenzoate CoA ligase offers additional insight into the molecular features that mediate substrate binding in adenylate-forming enzymes. This study provides the first biochemical characterization of a Box enzyme from LB400 and the first structural characterization of a Box enzyme from any organism, and further substantiates the concept of distinct roles for the two paralogous box pathways in LB400. [Copyright &y& Elsevier]

Burkholderia xenovorans LB400 harbours two paralogous copies of the recently discovered benzoate oxidation ( box) pathway. While both copies are functional, the paralogues are differentially regulated and flanked by putative transcriptional regulators from distinct families. The putative LysR-type transcriptional regulator (LTTR) adjacent to the megaplasmid-encoded box enzymes, Bxe_C0898, has been produced recombinantly in Escherichia coli and purified to homogeneity. Gel-filtration studies show that Bxe_C0898 is a tetramer in solution, consistent with previously characterized LTTRs. Bxe_C0898 crystallized with four molecules in the asymmetric unit of the P43212/ P41212 unit cell with a solvent content of 61.19%, as indicated by processing of the X-ray diffraction data. DNA-protection assays are currently under way in order to identify potential operator regions for this LTTR and to define its role in regulation of the box pathway. [ABSTRACT FROM AUTHOR]

Abstract: As a highly coveted precursor molecule, terephthalate (Tph) continues to be used extensively for the production of polyethylene Tph bottles, polyester films, and textile fibers worldwide. Based on its detrimental physiological effects, Tph is now recognized as a serious environmental pollutant. While amenable to biodegradation and, in fact, traditionally neutralized by aerobic microbiological processes, our current lack of understanding of the enzymatic degradation of Tph at the molecular level presents a major impediment in the development of robust bioremediation strategies. The biodegradation of Tph proceeds through a single metabolic intermediate (a cis-dihydrodiol), which is subsequently converted to the end product (protocatechuate) by a decarboxylating cis-dihydrodiol dehydrogenase (TphB). Using iodide single‐wavelength anomalous dispersion, we report the first structural characterization of TphB to 1.85Å resolution. Contrary to prior speculations, a fluorescent scan unambiguously shows that TphB coordinates Zn2+ and not Fe2+. The molecular architecture of TphB provides a rationale to the primary‐level divergence observed between TphB and other cis-dihydrodiol dehydrogenases while explaining its intriguingly close evolutionary clustering with non-dihydrodiol dehydrogenases belonging to the isocitrate/isopropylmalate family of enzymes. Sequence and structural analyses reveal a putative substrate-binding pocket proximal to the bound Zn2+. In silico substrate modeling in this putative binding pocket suggests a mechanistic sequence relying on H291, K295, and Zn2+ as core mediators of catalytic turnover. Overall, this study reveals novel structural and mechanistic insights into a decarboxylating cis-dihydrodiol dehydrogenase that mediates one of the two catalytic steps in the biodegradation of the environmental pollutant Tph. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]