Taxoid metabolism:: Taxoid 14β-hydroxylase is a cytochrome P450-dependent monooxygenase
Taxoid metabolism:: Taxoid 14β-hydroxylase is a cytochrome P450-dependent monooxygenase
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DOI：
10.1016/s0003-9861(03)00090-0
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发表时间：
2003-05-15
影响因子：
3.9
通讯作者：
Croteau, R
中科院分区：
文献类型：
--
作者：
Jennewein, S;Rithner, CD;Croteau, R
在红豆杉（红豆杉）细胞培养中，抗癌药物紫杉醇的产生通常伴随着带有14 -羟基的侧通路多氧类紫杉醇代谢物的形成。最近获得了几个新的半合成类taxoid中间体，可以筛选红豆杉细胞色素P450 cDNA克隆家族的14 - β -羟化酶和其他类taxoid加氧酶。候选细胞色素P450克隆在酵母中功能表达，并通过体内投喂放射性标记的5 α -乙酰氧基-10 β -羟基紫杉二烯和5 α，13 α -二羟基紫杉二烯进行检测。其中一个克隆高效特异地转化了5 α -乙酰氧基-10 - β -醇，而不是5 α，13 α -二醇，生成了一个极性更强的产品，具有类杉醇单乙酸三醇的色谱性质，并通过光谱手段证实了该产品的身份为5 α -乙酰氧基-10 β，14 β -二羟基杉二烯。从转化酵母制备的微粒体可以表征这种新的羟化酶，该酶与其他细胞色素P450类taxoid羟化酶相似，pH最适为7.5，类taxoid底物的K-m值约为50 muM。由于紫杉醇在c14位点未被取代，因此14- β -羟化酶不能存在于通往目标药物的途径上，而似乎负责将途径转移到14-羟基类紫杉醇，这是红豆杉细胞培养的主要代谢物。操纵这个羟化酶基因可以改变途径的方向，增加流向紫杉醇的通量，并可以制备13α。14 -羟基类taxoids作为新的治疗剂。(C) 2003 Elsevier Science（美国）版权所有。
The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K-m value for the taxoid substrate of about 50 muM. Because Taxol is unsubstituted at C 14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha.14beta-hydroxy taxoids as new therapeutic agents. (C) 2003 Elsevier Science (USA). All rights reserved.