二硫吡咯酮类抗生素中五元内酰胺环的生物合成研究

Dithiolopyrrolone antibiotics have broad-spectrum antibacterial activity and even possess anti-cancer property. It has also been reported that a series of chemical derivatives of Dithiolopyrrolone compounds could act as good prodrugs. However, the development of Dithiolopyrrolone for clinical utilization has been much impeded by its partially resolved biosynthesis mechanism, particularly the assembly logic of the bicyclic pyrrolinodithiole scaffold. In this context, we set out to investigate the enzymatic functions of the dehydrogenase HlmB, NRPS HlmE, and the condensation domain HlmL, which are proposed to be responsible for the biosynthesis of the lactam ring of the well-known Dithiolopyrrolone member, Holomycin. Through this project, novel enzymatic reactions involved in the biosynthesis of natural products will be unveiled, particularly the cyclization of lactam ring through a C-C bond formation catalyzed by an unusual Cy domain of HlmE. Furthermore, this project will also characterize the cascade reactions consisting of cysteine dehydrogenation, dipeptide condensation, and intramolecular cyclization, which leads to the assembly of the interesting pyrrolinodithiole scaffold. Therefore, our study will not only assist in understanding the assembly logic of the core skeleton of Dithiolopyrrolone, but also pave the way for future engineering of this group of promising antibiotics.

二硫吡咯酮类抗生素具有广谱抗菌活性，并能有效抑制肿瘤细胞的生长，其一系列衍生物被认为是优质的药物前体。然而，生物合成机制研究上的滞后很大程度上限制了该类药物的开发，其少见的双环内酰胺核心骨架部分的组装机制研究更是急需填补的空白。本项目拟以二硫吡咯酮代表性成员Holomycin作为模型，重点研究可能参与其吡咯酮环内酰胺组装的脱氢酶HlmB、多结构域NRPS蛋白HlmE和缩合结构域蛋白HlmL的酶学功能。本项目的实施一方面可以发现和证实天然产物生物合成过程中新的酶学功能和催化机制，特别是可能催化C-C键的形成完成分子内环化的一类特殊Cy结构域的催化机制；另一方面通过解析半胱氨酸的脱氢、二肽的缩合以及分子内环化这一级联酶催化反应，最终揭示吡咯酮环内酰胺的合成机制，从而填补二硫吡咯酮类抗生素核心骨架部分的生物合成研究空白，为进一步改造该类抗生素提供理论基础。

二硫吡咯酮类抗生素具有广谱抗菌活性和抗癌特性, 其生物合成研究一直是研究热点。但是二硫代吡咯酮生物合成机制的解析还未完善，特别是双环吡咯-二硫代吡咯支架的组装逻辑，阻碍了其临床应用的发展。本研究以硫黄素生物合成基因簇中的NRPS蛋白DtpB为例，研究肽键的形成机制。我们发现，腺苷化结构域不仅可以识别和腺苷化半胱氨酸，还可以辅助Cys-Cys二肽的形成。值得注意的是，本研究揭示了NRPS形成肽键的新机制，并扩展了腺苷化结构域的功能。