A Substrate-Permissive Vanadium Haloperoxidase in Type II Polyketide Biosynthesis Unlocks Non-Native Halogenation across Diverse Substrate Scaffolds

Abstract Halogenated compounds are prevalent in natural and synthetic molecules, profoundly affecting molecular stability, physicochemical properties, and biological activity. While enzymatic halogenation offers a sustainable and selective synthetic route, its broad application is limited by the narrow substrate scope of the known halogenases. Here, we identify and characterize a new secreted vanadium-dependent haloperoxidase (VHPO), XhnO11, from Streptomyces xinghaiensis S187, which catalyzes a critical chlorination step in the biosynthesis of the potent anticancer agent xinghaicarcin A. Through X-ray crystallography, molecular docking, and mutagenesis, we uncovered distinct mechanism features underlying bromination and revealed an unusually spacious active site in XhnO11. These structural characteristics enable remarkable substrate promiscuity, accommodating a diverse range of aromatic scaffolds, including anthracyclines, xanthones, flavonoids, and nitroaromatics. Furthermore, XhnO11 efficiently mediates mono-, di-, and multibromination of tyrosine, tyrosine-containing peptides, and tryptophan-derived substrates. Collectively, these findings expand the functional scope of VHPOs and establish XhnO11 as a versatile and broadly tolerant biocatalyst, facilitating mining of new halogenated natural products and providing a powerful tool for the selective functionalization of diverse bioactive molecules.