A Cytochrome P450 Involved in the Metabolism of Abietane Diterpenoids byPseudomonas abietaniphilaBKME-9

Abstract

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, P450 dit , 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 P450 dit expressed in Escherichia coli revealed that DhA binds to the enzyme and yields a type I binding spectrum with a K d of 0.4 M. These results indicate that P450 dit 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.

A Large Gene Cluster in Burkholderia xenovorans Encoding Abietane Diterpenoid Catabolism

“…This study demonstrates that LB400 is able to catabolize abietane diterpenoids as can several other bacteria (reviewed in references 24 and 26). However, in our initial growth tests, LB400 exhibited a much longer lag phase and lower growth rate (Table 2) than other characterized abietane-degrading bacteria (23,33). The reason for this lag phase is unclear, but the time seems too long to be solely due to genetic induction.…”

“…strain DhA-33, Zoolgea sp. strain DhA-35, and Pseudomonas abietaniphila BKME-9 (24,33). Further, after multiple passages on abietanes, LB400 cultures attained shorter lag phases and higher growth rates typical of other abietane degraders.…”

“…Analytes were derivatized by using diazomethane. GC electron impact MS of methyl ester derivatives was conducted as previously described (33), using an Agilent Technologies 6890N Network GC system equipped with an Agilent 5973 mass selective detector. National Institute of Standards and Technology MS Search (2.0) was used to analyze mass spectral data.…”

“…An additional catabolic capacity of LB400 was suggested when we previously identified 22 clustered genes in LB400 that are homologous to genes in the dit cluster of Pseudomonas abietaniphila BKME-9 (33), which encode abietane diterpenoid catabolism (20,21,33).…”

Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

“…Because of the narrow focus (PCB degradation) of past studies on LB400, most of its general physiology and lifestyle remain to be discovered. Since the initiation of the genome project, the interest in LB400 has broadened and has highlighted it as a versatile biodegrading soil organism metabolizing compounds containing single-carbon (C 1 ) groups (22,23), isoflavonoids (24), diterpenoids (25), and sulfonates (26).…”