Involvement of a cytochrome P450 monooxygenase in thaxtomin a biosynthesis by Streptomyces acidiscabies
Involvement of a cytochrome P450 monooxygenase in thaxtomin a biosynthesis by Streptomyces acidiscabies
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DOI：
10.1128/jb.184.7.2019-2029.2002
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发表时间：
2002-04-01
影响因子：
3.2
通讯作者：
Loria, R
中科院分区：
文献类型：
--
作者：
Healy, FG;Krasnoff, SB;Loria, R
紫杉素环二肽植物毒素的生物合成通过硫代模板机制以非核糖体的方式进行。两种氨基酸底物的酰基化、硫代酯化、N-甲基化和环化均由txAB编码的紫杉素合成酶催化。对酸链霉菌84.104的txtAB区3‘进行核苷酸序列分析，确定了一个编码P450单加氧酶基因家族同源基因的开放阅读框。认为紫杉素A的苯丙氨基羟化反应是由单加氧酶同系物催化的。利用整合型基因中断构建法对84.104菌株的ORF进行了突变，并对突变株的培养滤液提取物进行了紫杉素A脱羟基衍生物的检测。反相高效液相色谱和高效液相-质谱联用分析表明，突变株培养滤液提取物中的主要成分比紫杉素A的极性更弱且更小。电喷雾质谱图以及H-1和C-13核磁共振波谱的比较证实了该化合物的结构为12，15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl)，。克隆了泰索菌素A的双羟基类似物WC，并在大肠杆菌中表达并从细胞提取液中纯化了重组的6-His标记融合蛋白。在大肠杆菌中生产的TxtC表现出与细胞色素P450型血红蛋白类似的光谱特性，后者已转化为催化不活跃的P420形式。根据这些性质以及TxtC与其他已知的P450酶的高度相似性，我们得出结论：TxtC编码一种细胞色素P450型单加氧酶，是环二肽环化后羟化所必需的。
The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as H-1- and C-13-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated WC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.