Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-beta-farnesene
Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-beta-farnesene
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DOI：
10.1073/pnas.94.24.12833
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发表时间：
1997-11-25
影响因子：
11.1
通讯作者：
Croteau, R
中科院分区：
文献类型：
--
作者：
Crock, J;Wildung, M;Croteau, R
（E）-β-法呢烯是一种倍半萜类化学信息素，被植物和昆虫广泛用于交流。这种无环烯烃存在于薄荷（Mentha x piperita）的精油中，可以通过薄荷分泌腺细胞的无细胞提取物从法呢基二磷酸合成。从薄荷油腺文库中克隆了一个编码（E）-β-法呢烯合成酶的cDNA，并在大肠杆菌中表达。相应的合酶具有63.8kDa的推导大小，并且需要二价阳离子用于催化（Mg 2+的K-m约为150 μ M; Mn 2+的K-m约为7 μ M）。通过放射性-GC和GC-MS测定，由重组酶产生的倍半萜类化合物是（E）-β-法呢烯（85%）、（Z）-β-法呢烯（8%）和δ-杜松烯（5%），其天然C15底物法呢基二磷酸（K-m约为0.6 μ M; V-rel = 100）和Mg 2+作为辅因子，以及（E）-β-法呢烯（98%）和（Z）-β-法呢烯（2%）和Mn 2+作为辅因子（V-rel = 80）。用C-10类似物GDP作为底物（K-m = 1.5 μ M; V-rel = 3，Mg 2+作为辅因子），产生单萜柠檬烯（48%）、萜品油烯（15%）和月桂烯（15%）。
(E)-beta-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-beta-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (K-m for Mg2+ approximate to 150 mu M; K-m for Mn2+ approximate to 7 mu M). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-beta-farnesene (85%), (Z)-beta-farnesene (8%), and delta-cadinene (5%) with the native C15 substrate farnesyl diphosphate (K-m approximate to 0.6 mu M; V-rel = 100) and Mg2+ as cofactor, and (E)-beta-farnesene (98%) and (Z)-beta-farnesene (2%) with Mn2+ as cofactor (V-rel = 80). With the C-10 analog, GDP, as substrate (K-m = 1.5 mu M; V-rel = 3 with Mg2+ as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced.