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Structural and biophysical characterization of BoxC from Burkholderia xenovorans LB400: a novel ring-cleaving enzyme in the crotonase superfamily.

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.

SUBMITTER: Bains J

PROVIDER: S-EPMC2713537 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Structural and biophysical characterization of BoxC from Burkholderia xenovorans LB400: a novel ring-cleaving enzyme in the crotonase superfamily.

Bains Jasleen J Leon Rafael R Boulanger Martin J MJ

The Journal of biological chemistry 20090415 24

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 crot ...[more]

PMID: 19369256

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Project description:In this study, the gentisate and protocatechuate pathways in Burkholderia xenovorans LB400 were analyzed by genomic and functional approaches, and their role in 3-hydroxybenzoate (3-HBA) and 4-hydroxybenzoate (4-HBA) degradation was proposed. The LB400 genome possesses two identical mhbRTDHI gene clusters encoding the gentisate pathway and one mhbM gene encoding a 3-HBA 6-hydroxylase that converts 3-HBA into gentisate. The pca genes encoding the protocatechuate pathway and the pobA gene encoding the 4-HBA 3-monooxygenase that oxidizes 4-HBA into protocatechuate are arranged in gene clusters and single genes mainly at the minor chromosome, but also at the major chromosome and the megaplasmid. Strain LB400 was able to grow on gentisate, protocatechuate, 3-HBA and 4-HBA. Transcriptional analyses showed that the mhbD gene encoding the gentisate 1,2-dioxygenase was expressed during growth on 3-HBA, 4-HBA and gentisate, whereas the pcaG gene encoding the protocatechuate 3,4-dioxygenase was expressed only during growth on 4-HBA and protocatechuate. The mhbM gene encoding the 3-HBA 6-hydroxylase was transcribed in strain LB400 during growth on HBAs, gentisate, protocatechuate and glucose. The pobA gene encoding the 4-HBA 3-monooxygenase was expressed during growth on HBAs and glucose. 3-HBA- and 4-HBA-grown LB400 cells showed gentisate 1,2-dioxygenase activity, whereas protocatechuate 3,4-dioxygenase activity was observed only in 4-HBA-grown cells. The mhbR gene encoding a MarR-type transcriptional regulator that probably regulates the expression of the MhbT transporter, and the pcaQ and pcaR genes encoding LysR-type transcriptional regulators that regulate pcaHG and pcaIJBDC genes, respectively, were transcribed during growth on both HBAs, gentisate, protocatechuate and glucose, suggesting a basal constitutive expression. The results indicate active gentisate, protocatechuate, 3-HBA and 4-HBA catabolic pathways in B. xenovorans LB400 and suggest that 3-HBA is channeled exclusively through the gentisate route, whereas 4-HBA is funneled into the protocatechuate central pathway and potentially into the gentisate pathway.

Dataset Information

Structural and biophysical characterization of BoxC from Burkholderia xenovorans LB400: a novel ring-cleaving enzyme in the crotonase superfamily.

Publications

Structural and biophysical characterization of BoxC from Burkholderia xenovorans LB400: a novel ring-cleaving enzyme in the crotonase superfamily.

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