Liu Chengli, West Ryan C, Chen Muyuan, Cohn Whitaker, Wang George, Mandot Aryan M, Kim Selena, Cogan Dillon P
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA.
Division of CryoEM and Bioimaging, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025, USA.
bioRxiv. 2025 Jul 6:2025.07.05.663307. doi: 10.1101/2025.07.05.663307.
The rifamycin synthetase (RIFS) from the bacterium is a large (3.5 MDa) multienzyme system that catalyzes over 40 chemical reactions to generate a complex precursor to the antibiotic rifamycin B. It is considered a hybrid enzymatic assembly line and consists of an N-terminal nonribosomal peptide synthetase loading module followed by a decamodular polyketide synthase (PKS). While the biosynthetic functions are known for each enzymatic domain of RIFS, structural and biochemical analyses of this system from purified components are relatively scarce. Here, we examine the biosynthetic mechanism of RIFS through complementary crosslinking, kinetic, and structural analyses of its first PKS module (M1). Thiol-selective crosslinking of M1 provided a plausible molecular basis for previously observed conformational asymmetry with respect to ketosynthase (KS)-substrate carrier protein (CP) interactions during polyketide chain elongation. Our data suggest that C-terminal dimeric interfaces-which are ubiquitous in bacterial PKSs-force their adjacent CP domains to co-migrate between two equivalent KS active site chambers. Cryogenic electron microscopy analysis of M1 further supported this observation while uncovering its unique architecture. Single-turnover kinetic analysis of M1 indicated that although removal of C-terminal dimeric interfaces supported 2-fold greater KS-CP interactions, it did not increase the partial product occupancy of the homodimeric protein. Our findings cast light on molecular details of natural antibiotic biosynthesis that will aid in the design of artificial megasynth(et)ases with untold product structures and bioactivities.
来自该细菌的利福霉素合成酶（RIFS）是一个大型（3.5 MDa）多酶系统，催化40多个化学反应，生成抗生素利福霉素B的复杂前体。它被认为是一种混合酶组装线，由一个N端非核糖体肽合成酶加载模块和一个十模块聚酮合酶（PKS）组成。虽然已知RIFS每个酶结构域的生物合成功能，但对该系统从纯化组分进行的结构和生化分析相对较少。在这里，我们通过对其第一个PKS模块（M1）进行互补交联、动力学和结构分析，研究了RIFS的生物合成机制。M1的硫醇选择性交联为之前观察到的聚酮链延伸过程中酮合成酶（KS）-底物载体蛋白（CP）相互作用的构象不对称提供了一个合理的分子基础。我们的数据表明，在细菌PKS中普遍存在的C端二聚体界面迫使与其相邻的CP结构域在两个等效的KS活性位点腔室之间共同迁移。对M1的低温电子显微镜分析进一步支持了这一观察结果，同时揭示了其独特的结构。对M1的单轮动力学分析表明，虽然去除C端二聚体界面支持KS-CP相互作用增加2倍，但它并没有增加同二聚体蛋白的部分产物占有率。我们的研究结果揭示了天然抗生素生物合成的分子细节，这将有助于设计具有无数产物结构和生物活性的人工巨型合成酶。
