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J. Bacteriol. doi:10.1128/JB.01530-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Distinct roles for two CYP226 family cytochromes P450 in abietane diterpenoid catabolism by Burkholderia xenovorans LB400

Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

^* To whom correspondence should be addressed. Email: wmohn{at}interchange.ubc.ca.

	Abstract

The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. The cluster includes ditQ and ditU, predicted to encode cytochromes P450 of the poorly characterized CYP226A family. 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) versus succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s: DitU was only detected in the AbA-grown cells, whereas DitQ was expressed during growth both on DhA and on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither 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 DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (K_d = 0.98 ± 0.01 µM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate distinct roles of P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.