4-coumarate:coenzyme A ligase in hybrid poplar -: Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes
4-coumarate:coenzyme A ligase in hybrid poplar -: Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes
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DOI：
10.1104/pp.116.2.743
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发表时间：
1998-02-01
期刊：
影响因子：
7.4
通讯作者：
Douglas, CJ
中科院分区：
文献类型：
--
作者：
Allina, SM;Pri-Hadash, A;Douglas, CJ
酶4-香豆酸：辅酶A连接酶（4CL）在为苯丙素类天然产物生物合成提供活化硫酯底物中是重要的。我们测试了不同的杂交白杨（大果杨×美洲黑杨）组织的存在4CL异构体的快速蛋白质液相色谱法，并检测到至少三个4CL异构体。这些异构体共享类似的羟基肉桂酸底物利用概况，并对芥子酸都是无活性的，但不稳定的天然形式排除了广泛的进一步分析。4CL cDNA克隆被分离并分为两大类，其预测的氨基酸序列的86%相同。基因组Southern杂交表明，cDNA类代表两个白杨4CL基因，和北方杂交提供了证据，他们的差异表达。使用杆状病毒系统表达对应于这两个基因的重组酶。这两种重组蛋白质的底物利用概况彼此相似，并与天然白杨4CL亚型（4-香豆酸>阿魏酸>咖啡酸;没有芥子酸的转化）相似，除了两者对肉桂酸具有相对较高的活性。这些结果进行了讨论相对于4CL在苯丙烷代谢中的碳的分区的作用。
The enzyme 4-coumarate:coenzyme A ligase (4CL) is important in providing activated thioester substrates for phenylpropanoid natural product biosynthesis. We tested different hybrid poplar (Populos trichocarpa x Populus deltoides) tissues for the presence of 4CL isoforms by fast-protein liquid chromatography and detected a minimum of three 4CL isoforms. These isoforms shared similar hydroxycinnamic acid substrate-utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CL cDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity toward cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.