The Biosynthetic Origin of Irregular Monoterpenes in Lavandula ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF A NOVEL cis-PRENYL DIPHOSPHATE SYNTHASE GENE, LAVANDULYL DIPHOSPHATE SYNTHASE
The Biosynthetic Origin of Irregular Monoterpenes in Lavandula ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF A NOVEL cis-PRENYL DIPHOSPHATE SYNTHASE GENE, LAVANDULYL DIPHOSPHATE SYNTHASE
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DOI：
10.1074/jbc.m112.431171
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发表时间：
2013-03-01
影响因子：
4.8
通讯作者：
Mahmoud, Soheil S.
中科院分区：
文献类型：
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作者：
Demissie, Zerihun A.;Erland, Lauren A. E.;Mahmoud, Soheil S.
薰衣草精油主要由常规单萜组成，例如芳樟醇、1,8-桉树脑和樟脑。然而，它们还含有不规则单萜，包括薰衣草醇和乙酸薰衣草酯。尽管目前已知薰衣草中负责产生规则单萜的大多数基因，但尚未描述这些植物中催化不规则单萜生物合成的酶（包括薰衣草基二磷酸合酶（LPPS））。在这里，我们通过基于同源性的克隆策略报告了一种新型顺式异戊二烯基二磷酸合酶 cDNA（称为薰衣草 x 中间薰衣草基二磷酸合酶（LiLPPS））的分离和功能表征。 LiLPPS ORF编码305个氨基酸长的蛋白质，在大肠杆菌中表达，并通过镍-次氮基三乙酸亲和层析纯化重组蛋白。约34.5 kDa的细菌产生的蛋白质在体外特异性催化两个二甲基烯丙基二磷酸单元从头到中缩合成LPP，表观K-m和k(cat)值分别为208+/-12μM和0.1s(-1)。 LiLPPS 是一种同型二聚酶，具有 S 形饱和曲线，希尔系数为 2.7，表明其催化位点之间存在正向协同相互作用。 LiLPPS 可用于通过代谢工程调节植物中薰衣草醇及其衍生物的产生。
Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent K-m and k(cat) values of 208 +/- 12 mu M and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.