Revised Biosynthetic Pathway of Alnumycin Involves a Complex Interplay of Redox Reactions, Stereoisomerization, and an Atypical DUF6081 Isomerase

Abstract Alnumycin A is an antimicrobial natural product distinguished by an unusual 1,3-dioxane moiety attached to an aromatic polyketide core. While previous studies had established that d-ribose serves as the precursor to the 1,3-dioxane and its attachment to the aromatic ring system is catalyzed by a C-glycoside synthase, less is known about its subsequent transformation. Aln6 is a member of the domain of unknown function (DUF) 6081 protein family and was previously annotated as a cofactor-free oxidase. Herein, Aln6 is shown to catalyze a redox-neutral isomerization that converts the ribose moiety to a fused furan-dioxolane via an unprecedented rearrangement mechanism. Aln4 subsequently catalyzes an NADPH-dependent reductive ring-opening of the furan, setting the stage for nonenzymatic rearrangement of the dioxolane that produces the 1,3-dioxane structure and explains the stereochemical diversity of alnumycin A observed in vivo. Together, these findings not only revise the established view of the late-stage biosynthesis of alnumycin A but also redefine the chemistry of Aln6, expanding the catalytic repertoire of the DUF6081 family beyond canonical oxidative reactions.