Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1
Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1
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DOI：
10.1021/bi000644j
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发表时间：
2000-08-01
期刊：
影响因子：
2.9
通讯作者：
Ebizuka, Y
中科院分区：
文献类型：
--
作者：
Fujii, I;Mori, Y;Ebizuka, Y
编码 1,3,6,8-四羟基萘 (T4HN) 迭代 I 型聚酮合酶的炭疽菌 PKS1 基因在米曲霉中过表达。从转化体制备的无细胞提取物的SDS-PAGE分析显示出230000的强条带，其对应于推导的PKS 1蛋白的分子量。通过使用这种无细胞提取物，成功地证实了 T4HN 的体外合成，这是迄今为止检测到的真菌多芳环聚酮合酶活性的第一个例子。为了鉴定 T4HN 合成的起始单元，使用 C-14 标记的乙酰 CoA 和/或 C-14 标记的丙二酰 CoA 作为 T4HN 合酶反应的底物。观察到，即使在不存在乙酰辅酶A的情况下，C-14标记也仅从丙二酰辅酶A掺入到T4HN中，而不是从乙酰辅酶A掺入。该体外结果明确表明，丙二酰辅酶 A 在真菌聚酮合酶 PKS1 形成 T4HN 的过程中充当起始单元和延伸单元。
The Colletotrichum lagenarium PKS1 gene encoding iterative type I polyketide synthase of 1,3,6,8-tetrahydroxynaphthalene (T4HN) was overexpressed in Aspergillus oryzae. SDS-PAGE analysis of the cell-free extract prepared from the transformant showed an intense band of 230000 which corresponded to the molecular weight of the deduced PKS 1 protein. By using this cell-free extract, in vitro synthesis of T4HN was successfully confirmed as the first example of the fungal multi-aromatic ring polyketide synthase activity ever detected. To identify the starter unit fur T4HN synthesis, C-14-labeled acetyl CoA and/or C-14-labeled malonyl CoA were used as substrates for T4HN synthase reaction. Observed was the incorporation of C-14 label into T4HN solely from malonyl CoA even in the absence of acetyl CoA and not from acetyl CoA. This in vitro result unambiguously identified that malonyl CoA serves as the starter as well as extender units in the formation of T4HN by fungal polyketide synthase PKS1.