Regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha) species:: cDNA isolation, characterization, and functional expression of (-)-4S-limonene-3-hydroxylase and (-)-4S-limonene-6-hydroxylase
Regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha) species:: cDNA isolation, characterization, and functional expression of (-)-4S-limonene-3-hydroxylase and (-)-4S-limonene-6-hydroxylase
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DOI：
10.1006/abbi.1999.1298
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发表时间：
1999-08-01
影响因子：
3.9
通讯作者：
Croteau, R
中科院分区：
文献类型：
--
作者：
Lupien, S;Karp, F;Croteau, R
商品薄荷的环状单萜类化合物的氧化模式是通过区域特异性细胞色素P450催化的共同的烯烃前体(-)-4S-柠檬烯的羟化来确定的。在薄荷中，C3-烯丙基羟化反应得到(-)-反式-异戊二烯醇，而在留兰薄荷中，CG-烯丙基羟化反应产生(-)-反式香叶醇，从留兰香油腺体中纯化出微体柠檬烯-6-羟基酶，并利用均相酶的氨基酸序列设计了扩增片段为500bp的聚合酶链式反应引物。该非简并探针用于筛选留兰香油腺体的cDNA文库，从文库中分离出相应的全长cDNAs，并利用杆状病毒-Spotor tera系统进行功能表达，证实为CG-羟基酶。该探针还用于从薄荷油腺cDNA文库中分离出两个紧密相关的全长cDNAs，经功能表达证实为柠檬烯3-羟基酶。对这些区域特异性细胞色素P450单加氧酶的推导序列分析表明，这两种酶都带有一个典型的氨基末端膜锚定，与天然形式的微体位置一致，计算出的分子量分别为56,149(留兰薄荷)和56,560(薄荷)，并且在一级序列上非常相似(70%的同源性和85%的相似性)。这些区域化学上不同但密切相关的重组羟基酶及其相应基因的获得，为理解细胞色素P450底物结合和催化中的结构-功能关系提供了一个独特的模型系统，并为植物单萜生物合成途径的转基因操作提供了一种手段。(C)1999年学术出版社。
The oxygenation pattern of the cyclic monoterpenoids of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene, In peppermint (Mentha x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol, whereas in spearmint, CG-allylic hydroxylation leads to (-)-trans-carveol, The microsomal limonene-6-hydroxylase was purified from the oil glands of spearmint, and amino acid sequences from the homogeneous enzyme were used to design PCR primers with which a 500-bp amplicon was prepared. This nondegenerate probe was employed to screen a spearmint oil gland cDNA library from which the corresponding full-length cDNA was isolated and subsequently confirmed as the CG-hydroxylase by functional expression using the baculovirus-Spodoptera system. The probe was also utilized to isolate two closely related full-length cDNA species from a peppermint oil gland cDNA library which were confirmed as the limonene3-hydroxylase by functional expression as before. Deduced sequence analysis of these regiospecific cytochrome P450 monooxygenases indicates that both enzymes bear a typical amino-terminal membrane anchor, consistent with the microsomal location of the native forms, exhibit calculated molecular weights of 56,149 (spearmint) and about 56,560 (peppermint), and are very similar in primary sequence (70% identity and 85% similarity). The availability of these regiochemically distinct, yet very closely related, recombinant hydroxylases and their corresponding genes provides a unique model system for understanding structure-function relationships in cytochrome P450 substrate binding and catalysis, and a means for transgenic manipulation of monoterpene biosynthetic pathways in plants. (C) 1999 Academic Press.