Regioselectivity of taxoid-O-acetyltransferases:: heterologous expression and characterization of a new taxadien-5α-ol-O-acetyltransferase
Regioselectivity of taxoid-O-acetyltransferases:: heterologous expression and characterization of a new taxadien-5α-ol-O-acetyltransferase
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DOI：
10.1016/j.abb.2004.07.013
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发表时间：
2004-10-15
影响因子：
3.9
通讯作者：
Croteau, R
中科院分区：
文献类型：
--
作者：
Chau, MD;Walker, K;Croteau, R
除了抗癌药物紫杉醇之外，紫杉（Taxus）物种还产生大量其他紫杉烷二萜类化合物，其主要区别在于附加到紫杉烷核心上的许多羟基官能团上的酰基和芳酰基的类型;乙酸酯特别常见。紫杉醇在C10处带有乙酸酯，在C4处带有另一个乙酸酯，被认为来源于氧杂环丁烷环形成过程中C5乙酸酯官能团的分子内迁移，但许多其它天然存在的紫杉烷除C5和C10外还在C1、C2、C7、C9和C13处带有乙酸酯基团。编码紫杉烷5 α-O-乙酰转移酶（紫杉二烯-5 α-醇作为底物）和紫杉烷10 β-O-乙酰转移酶（10-脱乙酰浆果赤霉素III作为底物）的cDNA已从最近分离的红豆杉酰基/芳酰基转移酶克隆家族获得。为了探索其他乙酰化紫杉烷的起源，一组重组红豆杉酰基转移酶进行了研究与一系列的多羟基紫杉烷作为底物。从该研究中，鉴定了一种新的乙酰转移酶克隆（表示为TAX 19），其能够以与先前鉴定的5 α-O-乙酰转移酶（克隆TAX 1）相当的活性乙酰化紫杉二烯-5 α-醇。然而，当这两种重组酶与紫杉二烯三醇和四醇底物，他们表现出不同的区域特异性。TAX 1酶优先乙酰化C9和C10处的“北方”半球羟基，而TAX 19酶优先乙酰化C5和C13处的“东西”极点位置。TAX 1酶具有最低的K-M值与紫杉二烯-5a-of（早期途径代谢物）作为底物，具有高得多的KM值的多羟基化的紫杉烷底物，而TAX 19酶具有较低的KM值（比TAX 1转移酶）的所有紫杉烷底物测试。这些结果表明，TAX 1和TAX 19酰基转移酶可能在紫杉烷代谢的早期C5乙酰化步骤中起作用，并且TAX 19酰基转移酶，由于其对多羟基化紫杉烷的更广泛的特异性，也可能在代谢后期起作用，并负责许多其他乙酰化紫杉烷的产生。(C)2004年爱思唯尔公司All rights reserved.
In addition to the anticancer drug Taxol, yew (Taxus) species produce a large variety of other taxane diterpenoids which differ mainly in the type of acyl and aroyl groups appended to the many hydroxyl functions on the taxane core; acetate esters are particularly common. Taxol bears an acetate at C 10 and another at C4 thought to originate by intramolecular migration of a C5 acetate function in the process of oxetane ring formation, but many other naturally occurring taxoids bear acetate groups at C1, C2, C7, C9, and C13, in addition to C5 and C10. cDNAs encoding a taxoid 5alpha-O-acetyltransferase (taxadien-5alpha-ol as substrate) and a taxoid 10beta-O-acetyltransferase (10-deacetylbaccatin III as substrate) have been acquired from a recently isolated family of Taxus acyl/aroyltransferase clones. To explore the origins of other acetylated taxoids, the group of recombinant Taxus acyltransferases was investigated with a range of polyhydroxylated taxoids as substrates. From this survey, a new acetyltransferase clone (denoted TAX19) was identified that was capable of acetylating taxadien-5alpha-ol with activity comparable to that of the previously identified 5a-O-acetyltransferase (clone TAX1). However, when these two recombinant enzymes were presented with taxadien-triol and tetraol substrates, they exhibited different regiospecificities. The TAX1 enzyme preferentially acetylates the "northern" hemisphere hydroxyls at C9 and C10, whereas the TAX19 enzyme preferentially acetylates the "east-west" pole positions at C5 and C13. The TAX1 enzyme possesses the lowest K-M value with taxadien-5a-of (an early pathway metabolite) as substrate, with much higher KM values for the polyhydroxylated taxoid substrates, whereas the TAX19 enzyme possesses lower KM values (than the TAX1 transferase) for all taxoid substrates tested. These results suggest that both TAX1 and TAX19 acyltransferases may function at the early C5 acetylation step of taxoid metabolism, and that the TAX19 acyltransferase, because of its broader specificity for polyhydroxylated taxoids, may also function later in metabolism and be responsible for the production of many other acetylated taxoids. (C) 2004 Elsevier Inc. All rights reserved.