A cytochrome P450 involved in the metabolism of abietane diterpenoids by Pseudomonas abietaniphila BKME-9.
Diterpenoids are naturally occurring plant compounds which have pharmaceutical properties. We have sequenced a 10.4-kbp extension of the dit cluster in Pseudomonas abietaniphila BKME-9, containing genes involved in abietane diterpenoid biodegradation. The ditQ gene was found to encode a cytochrome P450 monooxygenase, P450dit, and to be homologous to the tdtD gene of Pseudomonas diterpeniphila A19-6a. Knocking out ditQ had little effect on growth of BKME-9 on abietic acid but severely impaired growth on dehydroabietic acid (DhA) and palustric acid (PaA), increasing doubling times from 3.8 to 15 h on DhA and from 5.6 to 18.5 h on PaA. A xylE transcriptional fusion showed that transcription of ditQ was induced by a range of diterpenoids. Substrate binding assays of P450dit expressed in Escherichia coli revealed that DhA binds to the enzyme and yields a type I binding spectrum with a Kd of 0.4 microM. These results indicate that P450dit is involved in the metabolism of DhA and PaA and are consistent with its putative role in converting DhA to 7-hydroxy-DhA. Finally, an amino acid sequence identity of greater than 72% and conserved gene arrangement support the hypothesis that the dit gene cluster of P. abietaniphila BKME-9 and the tdt cluster of P. diterpeniphila A19-6a contain functional homologues.
Smith
Daryl J
DJ
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Martin
Vincent J J
VJ
Mohn
William W
WW
D045784:Abietanes / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000502:physiology*; D005810:Multigene Family; D016366:Open Reading Frames; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism*
D045784:Abietanes; C013913:dehydroabietic acid; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System