A cytochrome P450 involved in the metabolism of abietane diterpenoids by Pseudomonas abietaniphila BKME-9
A cytochrome P450 involved in the metabolism of abietane diterpenoids by Pseudomonas abietaniphila BKME-9
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DOI：
10.1128/jb.186.11.3631-3639.2004
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发表时间：
2004-06-01
影响因子：
3.2
通讯作者：
Mohn, WW
中科院分区：
文献类型：
--
作者：
Smith, DJ;Martin, VJJ;Mohn, WW
二萜类化合物是天然存在的具有药物性质的植物化合物。我们已经测序了一个10.4 kbp的延伸的dit集群在假单胞菌abietaniphila BKME-9，包含基因参与松香烷二萜类生物降解。发现ditQ基因编码细胞色素P450单加氧酶P450（dit），并且与嗜二萜假单胞菌A19-6a的tdtD基因同源。敲除ditQ对BKME-9在枞酸上的生长几乎没有影响，但严重损害了脱氢枞酸（DhA）和长叶松酸（PaA）的生长，使DhA上的倍增时间从3.8小时增加到15小时，PaA上的倍增时间从5.6小时增加到18.5小时。xylE转录融合表明，ditQ的转录诱导的范围内的二萜类化合物。在大肠杆菌中表达的P450（dit）的底物结合测定揭示DhA结合酶并产生具有0.4 μ M的Kd的I型结合谱。这些结果表明P450（dit）参与DhA和PaA的代谢，并且与其在将DhA转化为7-羟基-DhA中的假定作用一致。最后，大于72%的氨基酸序列同一性和保守的基因排列支持了以下假设，即嗜冷杉假单胞菌BKME-9的dit基因簇和嗜二萜假单胞菌A19-6a的tdt基因簇含有功能同源物。
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 muM. 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.