Nitrogen deletion in the biosynthesis of HDAC-targeting anticancer drugs

Natural product biosynthetic pathways employ diverse enzymatic strategies for site-specific modifications of molecular scaffolds, tailoring their bioactivity. Here, we uncovered an unprecedented nitrogen deletion mechanism in the biosynthesis of the conserved pharmacophore of a natural depsipeptide histone deacetylase (HDAC) inhibitor family, exemplified by the clinical used anti-cancer drug romidepsin. Through combination of bioinformatics, genetic and biochemical characterization of their biosynthetic mechanism, we revealed novel catalytic functions within polyketide synthase and nonribosomal peptide synthetase assembly lines. Our findings demonstrate that a pair of multifunctional ketoreductase and dehydratase domains, coupled with a trans -acting phosphotransferase and a trans -acting flavin-dependent oxidoreductase, coordinate a series of transformations to remove the amino group introduced by the cysteine moiety in the pharmacophore of this HDAC inhibitor family. Deletion of this protruding amino group results in the formation of an aliphatic linker within the pharmacophore, mimicking the natural substrate of HDACs and conferring superior HDAC inhibitory activity of these compounds. Furthermore, we identified a novel condensation domain mediated a cryptic S -acylating protection mechanism in the biosynthesis of the conserved pharmacophore. Leveraging these insights, we established a highly efficient, one-pot enzymatic synthesis of the intact pharmacophore in its protected form, laying the groundwork for its scalable biocatalytic production. Our work expanded the mechanistic understanding of natural product site-specific editing enabled by novel functions of assembly line biosynthetic machineries. It also showcases how nature selectively sculpts molecular scaffolds to fine-tune bioactivity and diversify chemical space, offering new avenues for biocatalytic strategies in the development of this important family of HDAC inhibitors.