Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair
Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair
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DOI：
10.1073/pnas.1519695113
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发表时间：
2016-04-05
影响因子：
11.1
通讯作者：
Moore, Bradley S.
中科院分区：
文献类型：
--
作者：
El Gamal, Abrahim;Agarwal, Vinayak;Moore, Bradley S.
卤代吡咯（卤代吡咯）是生物活性细菌天然产物中常见的化学部分。含单卤代吡咯和二卤代吡咯的化合物的卤代吡咯部分源自保守机制，其中与载体蛋白结合的脯氨酸衍生的吡咯基首先被卤化，然后通过肽或聚酮化合物延伸进行加工。这种范式在珊瑚幼虫定居提示四溴吡咯 (1) 的海洋假交替单胞菌细菌生物合成过程中被打破，四溴吡咯是由脯氨酸衍生的羧酸盐被溴原子取代而产生的。为了了解 1 生物合成中脱羧溴化的分子基础，我们对两个假交替单胞菌基因组进行了测序，并鉴定了编码参与其完整生物合成的酶的保守四基因位点。通过使用纯化的酶对 1 的生物合成进行完全体外重建以及对各个生化步骤的生化询问，我们表明 1 中的所有四个溴原子都是通过单一黄素依赖性卤化酶 Bmp2 的作用安装的。吡咯的四溴化诱导硫酯酶介导的载体蛋白卸载反应，并激活生物合成中间体进行脱羧。通过与结构同源的卤化酶 Mpy16（在 marinopyrrole 生物合成中仅形成二卤代吡咯）对比，从卤化酶的高分辨率晶体结构中获得了对 Bmp2 四溴化活性的深入了解。所提出的 Bmp2 底物结合袋的结构引导诱变导致卤化催化程度降低。我们的研究为 1 的生物合成提供了生物遗传学基础，并为探究海洋（元）基因组中 1 的生物合成潜力奠定了坚实的基础。
Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono-and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate- binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta) genomes.