Cloning, functional expression, and characterization of CYP709C1, the first sub-terminal hydroxylase of long chain fatty acid in plants -: Induction by chemicals and methyl jasmonate
Cloning, functional expression, and characterization of CYP709C1, the first sub-terminal hydroxylase of long chain fatty acid in plants -: Induction by chemicals and methyl jasmonate
复制标题
DOI：
10.1074/jbc.m500918200
复制
发表时间：
2005-10-28
影响因子：
4.8
通讯作者：
Pinot, F
中科院分区：
文献类型：
--
作者：
Kandel, S;Morant, M;Pinot, F
我们克隆并鉴定了一种新的植物细胞色素CYP709C1，它属于P450家族，迄今为止除了与P450酶的脂肪酸代谢分支聚类外，还没有确定其功能。我们在这里展示了CYP709C1能够在-1和-2位置羟基化脂肪酸。从小麦cDNA文库中分离的全长cDNA在工程酵母菌株中重新编码并异源表达。对底物特异性的研究表明，CYP709C1代谢不同链长（C12至C18）和不饱和度的脂肪酸。CYP709C1是第一个被发现的能够催化C18脂肪酸亚末端羟基化的植物细胞色素P450。顺式9,10-环氧硬脂酸代谢效率最高，即K-m（app）为8 μ M， V-max（app）为328 nmol/min/nmol P450。这一点，再加上小麦拥有能够从油酸合成这种化合物的微粒体过加氧酶，强烈表明它是一种生理底物。羟基化脂肪酸与植物防御事件有关。我们假设CYP709C1可能通过产生这些化合物参与植物防御。这得到了以下观察结果的支持：(i)在胁迫激素茉莉酸甲酯处理的小麦幼苗微粒体中，9,10-环氧硬脂酸的亚末端羟基化被诱导（3小时后15倍）；（ii） CYP709C1在转录水平上被增强。CYP709C1转录本在安全剂萘二酸酐和苯巴比妥联合治疗后也有积累。这表明我们讨论过的CYP709C1可能具有解毒功能。
We cloned and characterized CYP709C1, a new plant cytochrome P450 belonging to the P450 family, that so far has no identified function except for clustering with a fatty acid metabolizing clade of P450 enzymes. We showed here that CYP709C1 is capable of hydroxylating fatty acids at the omega-1 and omega-2 positions. This work was performed after recoding and heterologous expression of a full-length cDNA isolated from a wheat cDNA library in an engineered yeast strain. Investigation on substrate specificity indicates that CYP709C1 metabolizes different fatty acids varying in their chain length (C12 to C18) and unsaturation. CYP709C1 is the first identified plant cytochrome P450 that can catalyze sub-terminal hydroxylation of C18 fatty acids. cis-9,10-Epoxystearic acid is metabolized with the highest efficiency, i.e. K-m(app) of 8 mu M and V-max(app) of 328 nmol/min/nmol P450. This, together with the fact that wheat possesses amicrosomal peroxygenase able to synthesize this compound from oleic acid, strongly suggests that it is a physiological substrate. Hydroxylated fatty acids are implicated in plant defense events. We postulated that CYP709C1 could be involved in plant defense by producing such compounds. This receives support from the observation that (i) sub-terminal hydroxylation of 9,10-epoxystearic acid is induced (15-fold after 3 h) in microsomes of wheat seedlings treated with the stress hormone methyl jasmonate and (ii) CYP709C1 is enhanced at the transcriptional level by this treatment. CYP709C1 transcript also accumulated after treatment with a combination of the safener naphthalic acid anhydride and phenobarbital. This indicates a possible detoxifying function for CYP709C1 that we discussed.