title: Distinct roles for two CYP226 family cytochromes P450 in abietane diterpenoid catabolism by Burkholderia xenovorans LB400.
abstract: The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 +/- 0.01 microM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, 2350 Health Science Mall, Vancouver, BC V6T 1Z3, Canada.
authors.forename: Daryl J
authors.initials: DJ
authors.lastname: Smith
authors.forename: Marianna A
authors.initials: MA
authors.lastname: Patrauchan
authors.forename: Christine
authors.initials: C
authors.lastname: Florizone
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
authors.forename: William W
authors.initials: WW
authors.lastname: Mohn
mesh_terms: D045784:Abietanes / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019117:Burkholderia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005810:Multigene Family / Q000235:genetics; D016254:Mutagenesis, Insertional; D010616:Phenanthrenes / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D019802:Succinic Acid / Q000378:metabolism
chemical_list: D045784:Abietanes; D001426:Bacterial Proteins; D010616:Phenanthrenes; C013913:dehydroabietic acid; D003577:Cytochrome P-450 Enzyme System; D019802:Succinic Acid; C023710:abietic acid