Monoterpene synthases from Grand fir (Abies grandis) - cDNA isolation, characterization, and functional expression of myrcene synthase, (-)(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase
Monoterpene synthases from Grand fir (Abies grandis) - cDNA isolation, characterization, and functional expression of myrcene synthase, (-)(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase
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DOI：
10.1074/jbc.272.35.21784
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发表时间：
1997-08-29
影响因子：
4.8
通讯作者：
Croteau, R
中科院分区：
文献类型：
--
作者：
Bohlmann, J;Steele, CL;Croteau, R
大冷杉（Abies grandis）已被开发为研究针叶树响应昆虫攻击或其他伤害而形成防御性油树脂的模型系统。油树脂的松节油部分是单萜（C-10）烯烃的复杂混合物，其中（-）-柠檬烯和（-）-α-和（-）-β-蒎烯是主要成分； (-)-柠檬烯和(-)-蒎烯合酶活性也在茎受伤时被诱导，基于相似性的克隆策略从堆积的茎cDNA文库中产生了三种新的cDNA物种，它们似乎编码三种不同的单萜合酶。在大肠杆菌中表达并以香叶基二磷酸作为底物进行酶测定后，随后通过手性相气相色谱和质谱法对萜产物进行分析表明这些序列编码(-)-柠檬烯合酶、月桂烯合酶和产生 α-蒎烯和 β-蒎烯的 (-)-蒎烯合酶。在性质和反应立体化学方面，重组酶类似于伤口诱导的杉木茎中相应的天然单萜合酶。推导的氨基酸序列表明柠檬烯合酶的长度为637个残基(73.5kDa)，月桂烯合酶的长度为627个残基(72.5kDa)，蒎烯合酶的长度为628个残基(71.5kDa)；所有这些单萜合酶似乎都被翻译为带有氨基末端质体靶向序列的前蛋白。序列比较显示，这些来自大冷杉的单萜合酶与来自针叶树的倍半萜 (C-15) 合酶和二萜 (C-20) 合酶比来自被子植物物种的其他单萜合酶更相似。裸子植物现存的单萜、倍半萜和二萜合酶之间的这种相似性令人惊讶，因为此类酶的功能多样化被认为发生在 3 亿多年前。使用源自三种单萜合酶 cDNA 的探针进行 RNA 印迹杂交，证明了伤口诱导的单萜合酶转录物积累。编码这些单萜合酶的 cDNA 物种的可用性有助于了解针叶树中油树脂形成的调节，并最终允许对这种防御性分泌进行转基因操作，以增强对昆虫的抵抗力。这些 cDNA 还提供了用于定义生成无环、单环和双环烯烃产物的这组催化剂中的结构-功能关系的工具。
Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury, The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C-10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding, A similarity based cloning strategy yielded three new cDNA species from a mounded stem cDNA library that appeared to encode three distinct monoterpene synthases, After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C-15) synthases and diterpene (C-20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA blot hybridization using probes derived from the three monoterpene synthase cDNAs. The availability of cDNA species encoding these monoterpene synthases mill allow an understanding of the regulation of oleoresin formation in conifers and will ultimately permit the transgenic manipulation of this defensive secretion to enhance resistance to insects. These cDNAs also furnish tools for defining structure-function relationships in this group of catalysts that generate acyclic, monocyclic, and bicyclic olefin products.