cDNA cloning, functional expression and characterization of ent-copalyl diphosphate synthase from Scoparia dulcis L.
cDNA cloning, functional expression and characterization of ent-copalyl diphosphate synthase from Scoparia dulcis L.
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DOI：
10.1016/j.plantsci.2005.05.028
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发表时间：
2005-10-01
期刊：
影响因子：
5.2
通讯作者：
Hayashi, T
中科院分区：
文献类型：
--
作者：
Nakagiri, T;Lee, JB;Hayashi, T
黄荆（玄参科）产生几类二萜，其被提出通过顺-柯巴基二磷酸（syn-CDP）生物合成。另一方面，已报道赤霉素通过ent-CDP生物合成并充当生长调节剂。因此，S. dulcis应该具有至少两个编码内-和顺-柯巴基二磷酸脱氢酶（CPS）的酶基因，所述酶基因催化GGDR的环化。使用基于植物CPS的共有序列的简并引物的RT-PCR方法允许分离显示与植物CPS高度相似性的cDNA片段。利用RACE方法获得了该基因的全长cDNA，ORF为2475 bp，编码825个氨基酸残基。在大肠杆菌中表达的重组蛋白可催化香叶基香叶基二磷酸（GGDP）环化生成ent-CDP。因此，发现该酶是ent-CPS，我们将其命名为SdECPS。该酶在30 ℃和pH 7.4下表现出最佳活性，并且需要Mg 2+离子作为金属辅因子。SdECPS的N端含有一个叶绿体定位信号序列，其截短蛋白的CPS活性高于天然酶，而N端111个氨基酸残基的缺失显著降低了该酶的活性。(c)2005爱思唯尔爱尔兰有限公司保留所有权利。
Scoparia dulcis L. (Scrophulariaceae) produces several classes of diterpenes, which are proposed to be biosynthesized via syn-copalyl diphosphate (syn-CDP). On the other hand, gibberellin has been reported to be biosynthesized via ent-CDP and acts as a growth regulator. Therefore, S. dulcis should possess at least two enzymatic genes encoding ent- and syn-copalyl diphosphate synthases (CPSs) which catalyze cyclizations of GGDR An RT-PCR method using degenerate primers based on the consensus sequences of plant CPSs allowed the isolation of a cDNA fragment showing a high similarity to plant CPSs. The full-length cDNA obtained using the rapid amplification of cDNA ends (RACE) method contained 2475 bp ORF encoding 825 amino acid residues. The recombinant protein expressed in Escherichia coli catalyzed cyclization of geranylgeranyl diphosphate (GGDP) to ent-CDP. Therefore, the enzyme was found to be ent-CPS, which we named SdECPS. The enzyme showed optimum activity at 30 degrees C and pH 7.4 and required Mg2+ ion as metallic co-factor. In addition, SdECPS was found to bear an N-terminal chloroplast targeting signal sequence and its truncated protein showed higher CPS activity than the native enzyme, while deletion of more than 111 amino acid residues of N-terminal sequence significantly reduced this, activity. (c) 2005 Elsevier Ireland Ltd. All rights reserved.