Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Department of Natural Product Biotechnology, Helmholtz Centre for Infection Research (HZI) and Department of Pharmacy at Saarland University, Campus E8.1, 66123 Saarbrücken, Germany.
Chair of Technical Biochemistry, Technische Universität Dresden, Bergstraße 66, 01069 Dresden, Germany.
J Am Chem Soc. 2024 Jun 12;146(23):16203-16212. doi: 10.1021/jacs.4c04115. Epub 2024 Jun 3.
Baeyer-Villiger monooxygenases (BVMOs) play crucial roles in the core-structure modification of natural products. They catalyze lactone formation by selective oxygen insertion into a carbon-carbon bond adjacent to a carbonyl group (Baeyer-Villiger oxidation, BVO). The homologous bacterial BVMOs, BraC and PxaB, thereby process bicyclic dihydroindolizinone substrates originating from a bimodular nonribosomal peptide synthetase (BraB or PxaA). While both enzymes initially catalyze the formation of oxazepine-dione intermediates following the identical mechanism, the final natural product spectrum diverges. For the pathway involving BraC, the exclusive formation of lipocyclocarbamates, the brabantamides, was reported. The pathway utilizing PxaB solely produces pyrrolizidine alkaloids, the pyrrolizixenamides. Surprisingly, replacing within the pyrrolizixenamide biosynthetic pathway by does not change the product spectrum to brabantamides. Factors controlling this product selectivity have remained elusive. In this study, we set out to solve this puzzle by combining the total synthesis of crucial pathway intermediates and anticipated products with in-depth functional in vitro studies on both recombinant BVMOs. This work shows that the joint oxazepine-dione intermediate initially formed by both BVMOs leads to pyrrolizixenamides upon nonenzymatic hydrolysis, decarboxylative ring contraction, and dehydration. Brabantamide biosynthesis is enzyme-controlled, with BraC efficiently transforming all the accepted substrates into its cognate final product scaffold. PxaB, in contrast, shows only considerable activity toward brabantamide formation for the substrate analog with a natural brabantamide-type side chain structure, revealing substrate-controlled product selectivity.
Baeyer-Villiger 单加氧酶（BVMOs）在天然产物的核心结构修饰中发挥着关键作用。它们通过选择性地将氧插入到羰基基团相邻的碳-碳键中，催化内酯的形成（Baeyer-Villiger 氧化，BVO）。同源的细菌 BVMOs，BraC 和 PxaB，从而处理来源于双模块非核糖体肽合成酶（BraB 或 PxaA）的双环二氢吲哚嗪酮底物。虽然这两种酶最初都遵循相同的机制，首先催化恶唑嗪-二酮中间体的形成，但最终的天然产物谱却有所不同。对于涉及 BraC 的途径，仅报道了脂环氨基甲酸酯，即 brabantamides 的专一形成。利用 PxaB 的途径仅产生吡咯里西啶生物碱，即 pyrrolizixenamides。令人惊讶的是，在 pyrrolizixenamide 生物合成途径中用 取代 ，并不会将产物谱改变为 brabantamides。控制这种产物选择性的因素仍然难以捉摸。在这项研究中，我们通过将关键途径中间体和预期产物的全合成与对两种重组 BVMOs 的深入功能体外研究相结合，旨在解决这个难题。这项工作表明，最初由两种 BVMOs 形成的联合恶唑嗪-二酮中间体在非酶水解、脱羧环缩合和脱水作用下导致产生 pyrrolizixenamides。Brabantamide 生物合成是酶控的，BraC 有效地将所有接受的底物转化为其同源最终产物支架。相比之下，PxaB 仅对具有天然 brabantamide 型侧链结构的底物类似物表现出相当大的形成 brabantamide 的活性，揭示了底物控制的产物选择性。
