Molecular cloning of a taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli.
Molecular cloning of a taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli.
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DOI：
10.1006/abbi.1999.1609
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发表时间：
2000-02
影响因子：
3.9
通讯作者：
Kevin D. Walker;A. Schoendorf;R. Croteau
中科院分区：
文献类型：
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作者：
Kevin D. Walker;A. Schoendorf;R. Croteau
从茉莉酸甲酯诱导的红豆杉细胞中分离得到了催化紫杉醇生物合成第三步的Taxa-4(20)，11(12)-dien-5α-ol-O-乙酰基转移酶，并对其进行了部分纯化和鉴定(K.Walker，R.E.B.Ketchum，M.Hezari，D.Gatfield，M.Golenowski，A.Barthol和R.Croteau，Arch.生物化学。生物群落。364、273-279 1999)。一种改进的纯化方法允许对该酶进行内部氨基酸微测序，根据该微序列设计引物并用于扩增转乙酰基酶基因特异性片段。用这个900bp的放射性标记扩增子作为杂交探针，筛选从诱导红豆杉细胞中分离的Poly(A)(+)RNA构建的cDNA文库，从文库中获得了全长的转乙酰基酶序列。将pCWori(+)克隆在大肠杆菌JM109细胞中表达，经放射化学分析和毛细管气相色谱-质谱联用验证乙酰化产物后，获得了功能酶。全长DNA有一个1317个核苷酸的开放阅读框，对应于439个残基的推导氨基酸序列，与重组转乙酰基酶在性质上相似的天然酶的蛋白水解段具有很高的序列同源性。与可操作的天然酶的大小一致，DNA似乎编码一个分子量为49,079的单体蛋白，它没有N-末端细胞器靶向信息。紫杉烯-5α-醇-O-乙酰基转移酶与少数已知的植物来源的酰基转移酶的序列比较表明，这些酶之间有显著的相似性(-67%)。紫杉烯-5α-乙酸酯有效地转化为紫杉醇途径的进一步羟化中间产物，证实了这一酰化步骤的重要性，并表明该紫杉醇转乙酰基酶是提高紫杉醇产量的基因操作的重要靶点。
The taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase which catalyzes the third step of Taxol biosynthesis has been isolated from methyl jasmonate-induced Taxus cells, and partially purified and characterized (K. Walker, R. E. B. Ketchum, M. Hezari, D. Gatfield, M. Golenowski, A. Barthol, and R. Croteau, Arch. Biochem. Biophys. 364, 273-279 1999). A revised purification method allowed internal amino acid microsequencing of the enzyme, from which primers were designed and employed to amplify a transacetylase gene-specific fragment. This radiolabeled, 900-bp amplicon was used as a hybridization probe to screen a cDNA library constructed from poly(A)(+) RNA isolated from induced Taxus cells, from which a full-length transacetylase sequence was obtained. Expression of this clone from pCWori(+) in Escherichia coli JM109 cells yielded the functional enzyme, as determined by radiochemical assay and combined capillary gas chromatographic-mass spectrometric verification of the acetylated product. The full-length DNA has an open-reading frame of 1317 nucleotides corresponding to a deduced amino acid sequence of 439 residues that exhibits high sequence identity to the proteolytic fragments of the native enzyme, which the recombinant transacetylase resembles in properties. Consistent with the size of the operationally soluble native enzyme, the DNA appears to encode a monomeric protein of molecular weight 49,079 that bears no N-terminal organellar targeting information. Sequence comparison of the taxadien-5alpha-ol-O-acetyl transferase with the few other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (64-67%). The efficient conversion of taxadien-5alpha-yl acetate to further hydroxylated intermediates of the Taxol pathway confirms the significance of this acylation step and suggests this taxadienol transacetylase to be an important target for genetic manipulation to improve Taxol production.