Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis
Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis
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DOI：
10.1073/pnas.0506964102
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发表时间：
2005-09-27
影响因子：
11.1
通讯作者：
Walsh, CT
中科院分区：
文献类型：
--
作者：
Dorrestein, PC;Yeh, E;Walsh, CT
抗真菌天然产物pyoluerecrems in含有4，5-二氯吡咯部分。杂芳环中二氯化的时机现在显示发生在脯氨酸通过硫酯键连接到载体蛋白PltL并酶促去饱和为吡咯基-S-PItL之后。令人惊讶的是，FADH（2）依赖性卤化酶PltA在环的两个位置催化氯化，产生5-氯吡咯基-S-PltL中间体，然后产生4，5-二氯吡咯基-S-PltL产物。PltA活性严格依赖于异源黄素还原酶，该酶使用NAD（P）H产生FADH（2）。电喷雾电离-傅立叶变换MS检测到5个共价中间体连接到11-kDa的载体蛋白PltL。串联MS定位载体蛋白支架上的共价修饰位点。水解产物的HPLC分析与在杂芳环的5位然后4位的区域特异性氯化一致。提出了二氯化的机制，涉及形成FAD-4a-OCI中间体，用于被杂芳族吡咯部分的富电子C4和C5捕获。
The antifungal natural product pyoluteorin contains a 4,5-dichloropyrrole moiety. The timing of dichlorination in the heteroaromatic ring is now shown to occur after proline is tethered by thioester linkage to the carrier protein PltL and enzymatically desaturated to the pyrrolyl-S-PItL. Surprisingly, the FADH(2)-dependent halogenase PltA catalyzes chlorination at both positions of the ring, generating the 5-chloropyrrolyl-S-PItL intermediate and then the 4,5-dichloropyrrolyi-S-PltL product. PltA activity strictly depends on a heterologous flavin reductase that uses NAD(P)H to produce FADH(2). Electrospray ionization-Fourier transform MS detected five covalent intermediates attached to the 11-kDa carrier protein PltL. Tandem MS localized the site of covalent modification on the carrier protein scaffold. HPLC analysis of the hydrolyzed products was consistent with the regiospecific chlorination at position 5 and then position 4 of the heteroaromatic ring. A mechanism for dichlorination is proposed involving formation of a FAD-4a-OCI intermediate for capture by the electron-rich C4 and C5 of the heteroaromatic pyrrole moiety.