Regiodivergent NC and NN Aryl Coupling Reactions of Indoloterpenes and Cycloether Formation Mediated by a Single Bacterial Flavoenzyme

Angewandte Chemie International Edition
2013.0

Abstract

Indoloterpenoid natural products play an eminent role as drugs, but bacterial pathways were virtually unknown until recent discovery of indolosesquiterpenes like xiamycin. Previous analyses of the xiamycin biosynthesis gene cluster revealed N-C/N-N coupled dimers (bixiamycins) whose biosynthesis remained enigmatic. Herein, we report the discovery and full characterization of a series of highly regiodivergent, N-C and N-N aryl-coupled xiamycin dimers, reveal their potent antibacterial activities (N-N coupled dimers most active, with no cytotoxicity against tumor cell lines), and show that a single flavoprotein (XiaH) not only mediates diverse aryl couplings but also ether formation (for oxiamycin). We support a radical-based mechanism by a biomimetic synthesis of xiamycin derivatives. Through heterologous expression of the entire xia gene cluster, we identified bixiamycins; structural characterization via HPLC-HRMS and NMR revealed their xiamycin backbone and atropodiastereomeric pairs. Mutational analyses and heterologous expression confirmed XiaH (a FAD-dependent flavoprotein) is essential for dimer formation and oxiamycin production. Bioinformatic and phylogenetic analyses placed XiaH in the family of flavin adenine dinucleotide (FAD) dependent aromatic ring hydroxylases. Biomimetic synthesis using benzoyl peroxide (a radical starter) of bixiamycins and oxiamycin validated the radical mechanism. Our findings highlight XiaH's multifunctionality in generating structural diversity and potent antibiotics, suggesting its use as a novel biocatalyst for bixiamycin analogues.

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