Biosynthetic studies of aziridine formation in azicemicins.
Biosynthetic studies of aziridine formation in azicemicins.
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DOI：
10.1021/ja907307h
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发表时间：
2009-12-23
影响因子：
15
通讯作者：
Liu, Hung-wen
中科院分区：
文献类型：
--
作者：
Ogasawara, Yasushi;Liu, Hung-wen
阿奇霉素是由放线菌Kibdelosporangium sp.产生的一种广古环素类抗生素。MJ126-NF4含有连接在聚酮核心上的氮杂环。用[1-13C]乙酸酯或[1，2-13C2]乙酸酯喂养实验表明，绞股蓝环素骨架是由II型聚酮合成酶生物合成的。用氚标记的氨基酸进行的同位素示踪实验表明，天冬氨酸是氮杂环丙氨酸的前体。随后的克隆和测序工作导致了跨度约50kbp的阿奇米星(Azic)基因簇的鉴定。该簇含有典型的II型聚酮合成基因。该簇还包含两个腺酰转移酶、一个脱羧酶、一个额外的酰基载体蛋白(ACP)和几种加氧酶的基因。基于这些基因的指定功能，现在可以提出在阿奇米星中形成氮杂环的可能途径。为了获得对所提出的生物合成途径的支持，我们过表达了两个编码腺苷转移酶的基因，并对所产生的蛋白进行了纯化。酶分析表明，其中一种腺苷转移酶能特异性识别天冬氨酸，除了喂养实验外，还提供了强有力的证据，证明天冬氨酸是氮杂环丙烷部分的前体。本文报道的结果为未来氮杂环丙啶生物合成和组装的生物化学研究奠定了基础。
The azicemicins, which are angucycline-type antibiotics produced by the actinomycete, Kibdelosporangium sp. MJ126-NF4, contain an aziridine ring attached to the polyketide core. Feeding experiments using [1-13C]acetate or [1,2-13C2] acetate indicated that the angucycline skeleton is biosynthesized by a type II polyketide synthase. Isotope-tracer experiments using deuterium-labeled amino acids revealed that aspartic acid is the precursor of the aziridine moiety. Subsequent cloning and sequencing efforts led to the identification of the azicemicin (azic) gene cluster spanning ~50 kbp. The cluster harbors genes typical for type II polyketide synthesis. Also contained in the cluster are genes for two adenylyl transferases, a decarboxylase, an additional acyl carrier protein (ACP), and several oxygenases. On the basis of the assigned functions of these genes, a possible pathway for aziridine ring formation in the azecimicins can now be proposed. To obtain support for the proposed biosynthetic pathway, two genes encoding adenylyltransferases were overexpressed and the resulting proteins were purified. Enzyme assays showed that one of the adenylyltransferases specifically recognizes aspartic acid, providing strong evidence, in addition to the feeding experiments, that aspartate is the precursor of the aziridine moiety. The results reported herein set the stage for future biochemical studies of aziridine biosynthesis and assembly.
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