An Alpha/beta-hydrolase Fold Protein in the Biosynthesis of Thiostrepton Exhibits a Dual Activity for Endopeptidyl Hydrolysis and Epoxide Ring Opening/macrocyclization
An Alpha/beta-hydrolase Fold Protein in the Biosynthesis of Thiostrepton Exhibits a Dual Activity for Endopeptidyl Hydrolysis and Epoxide Ring Opening/macrocyclization
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硫链丝菌素生物合成中的α/β-水解酶折叠蛋白表现出内肽基水解和环氧化物开环/大环化的双重活性
DOI：
10.1073/pnas.1612607113
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发表时间：
2016
影响因子：
11.1
通讯作者：
Wen Liu
中科院分区：
文献类型：
--
作者：
Qingfei Zheng;Shoufeng Wang;Panpan Duan;Rijing Liao;D;an Chen;Wen Liu
硫链丝菌肽 (TSR) 是一种典型的双大环硫肽抗生素，由基因编码的前体肽进行复杂的翻译后修饰而产生，在硫肽特征框架的侧环系统内拥有喹哪啶酸 (QA) 部分。专注于 QA 部分的选择性工程，即通过氟化或甲基化，我们最近设计并生物合成了生物活性更高的 TSR 类似物。使用这些类似物作为化学探针，我们发现了 TSR 型硫肽的一种不寻常的间接机制，除了直接靶向细菌核糖体之外，它还能够通过宿主自噬诱导来对抗细胞内病原体。在此，我们报告了 6'-氟-7', 8'-环氧-TSR 的积累，这是制备类似物 6'-氟-TSR 的关键中间体。这一意外发现揭示了 TSR 成熟过程，其中涉及 TsrI（一种 α/β-水解酶折叠蛋白）的不寻常双重活性，用于在侧环系统构建过程中级联 C-N 键裂解和形成。 TsrI 的这两个功能依赖于相同的催化三联体 Ser72-His200-Asp191，它首先介导在残基 Met-1 和 Ile1 之间选择性发生的内肽基水解，以去除前导肽，然后触发环氧化物环打开，以区域和立体特异性方式关闭包含 QA 的侧环系统。前一个反应可能需要形成酰基-Ser72 酶中间体；相反，后者独立于Ser72。因此，QA 的 C-6' 氟化降低了环氧化物中间体的反应性，从而可以解剖 TsrI 相关的酶促过程，该过程快速进行，通常在 TSR 生物合成过程中很难实现。
Thiostrepton (TSR), an archetypal bimacrocyclic thiopeptide antibiotic that arises from complex posttranslational modifications of a genetically encoded precursor peptide, possesses a quinaldic acid (QA) moiety within the side-ring system of a thiopeptide-characteristic framework. Focusing on selective engineering of the QA moiety, i.e., by fluorination or methylation, we have recently designed and biosynthesized biologically more active TSR analogs. Using these analogs as chemical probes, we uncovered an unusual indirect mechanism of TSR-type thiopeptides, which are able to act against intracellular pathogens through host autophagy induction in addition to direct targeting of bacterial ribosome. Herein, we report the accumulation of 6′-fluoro-7′, 8′-epoxy-TSR, a key intermediate in the preparation of the analog 6′-fluoro-TSR. This unexpected finding led to unveiling of the TSR maturation process, which involves an unusual dual activity of TsrI, an α/β-hydrolase fold protein, for cascade C-N bond cleavage and formation during side-ring system construction. These two functions of TsrI rely on the same catalytic triad, Ser72-His200-Asp191, which first mediates endopeptidyl hydrolysis that occurs selectively between the residues Met-1 and Ile1 for removal of the leader peptide and then triggers epoxide ring opening for closure of the QA-containing side-ring system in a regio- and stereo-specific manner. The former reaction likely requires the formation of an acyl-Ser72 enzyme intermediate; in contrast, the latter is independent of Ser72. Consequently, C-6′ fluorination of QA lowers the reactivity of the epoxide intermediate and, thereby, allows the dissection of the TsrI-associated enzymatic process that proceeds rapidly and typically is difficult to be realized during TSR biosynthesis.