Partial purification and characterization of acetyl coenzyme A:: Taxa-4(20),11(12)-dien-5α-ol O-acetyl transferase that catalyzes the first acylation step of Taxol biosynthesis
Partial purification and characterization of acetyl coenzyme A:: Taxa-4(20),11(12)-dien-5α-ol O-acetyl transferase that catalyzes the first acylation step of Taxol biosynthesis
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DOI：
10.1006/abbi.1999.1125
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发表时间：
1999-04-15
影响因子：
3.9
通讯作者：
Croteau, R
中科院分区：
文献类型：
--
作者：
Walker, K;Ketchum, REB;Croteau, R
Taxa-4(20),11 (12)-dien-5 α-ol 的乙酰化被认为是紫杉醇生物合成的第三个具体步骤，该步骤先于紫杉烷核的进一步羟基化。在用茉莉酸甲酯诱导的加拿大红豆杉和东北红豆杉细胞的提取物中证明了可操作的可溶性乙酰辅酶A：紫杉二烯醇-O-乙酰基转移酶，以产生紫杉醇。该反应依赖于共底物和活性酶，并且通过放射色谱和 GC-MS 分析鉴定了该乙酰转移酶的产物。确定诱导细胞培养物中乙酰转移酶出现的时间过程后，通过阴离子交换、疏水相互作用和固定化辅酶 A 树脂上的亲和层析相结合，部分纯化可操作的可溶性酶。该乙酰转移酶的最适pI和pH分别为4.7和9.0，通过凝胶渗透色谱法测定的分子量约为50,000。该酶对两种共底物均表现出高选择性和高亲和力，对紫杉二烯醇和乙酰 CoA 的 K-m 值分别为 4.2 和 5.5 μM。该酶不会乙酰化更高级的紫杉醇前体、10-脱乙酰浆果赤霉素 III 或浆果赤霉素 III。这种乙酰基转移酶对单价和二价金属离子不敏感，仅受对羟基汞苯甲酸酯、N-乙基马来酰亚胺和辅酶 A 的微弱抑制，并且在一般特性上与已检查的少数其他高等植物来源的 O-乙酰基转移酶相似。 (C) 1999 年学术出版社。
The acetylation of taxa-4(20),11 (12)-dien-5 alpha-ol is considered to be the third specific step of Taxol biosynthesis that precedes further hydroxylation of the taxane nucleus. An operationally soluble acetyl CoA:taxadienol-O-acetyl transferase was demonstrated in extracts of Taxus canadensis and Taxus cuspidata cells induced with methyl jasmonate to produce Taxol. The reaction was dependent on both cosubstrates and active enzyme, and the product of this acetyl transferase was identified by radiochromatographic and GC-MS analysis. Following determination of the time course of acetyl transferase appearance in induced cell cultures, the operationally soluble enzyme was partially purified by a combination of anion exchange, hydrophobic interaction, and affinity chromatography on immobilized coenzyme A resin. This acetyl transferase has a pI and pH optimum of 4.7 and 9.0, respectively, and a molecular weight of about 50,000 as determined by gel permeation chromatography. The enzyme shows high selectivity and high affinity for both cosubstrates, with K-m values of 4.2 and 5.5 mu M for taxadienol and acetyl CoA, respectively. The enzyme does not acetylate the more advanced Taxol precursors, 10-deacetylbaccatin III or baccatin III. This acetyl transferase is insensitive to monovalent and divalent metal ions, is only weakly inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and coenzyme A, and resembles in general properties the few other O-acetyl transferases of higher plant origin that have been examined. (C) 1999 Academic Press.