Binuclear Copper‐Dependent Oxidative Enzymes Involved in Fungal Natural Product Modifications

ABSTRACT Binuclear copper‐dependent oxidative enzymes (BiNCOs), previously annotated as domain of unknown function 3328 (DUF3328) or UstYa homologs, are involved in the oxidative modification during fungal natural product biosynthesis. Although this family was discovered over a decade ago, the biochemical properties, especially cofactor dependency, remained unsolved until the recent characterization of the C(sp 3 )H halogenase ApnU. Utilizing a protein refolding strategy to obtain soluble and functional enzymes, the unexpected copper‐dependency of ApnU was demonstrated. Subsequent studies with members in this family, including macrocyclase AprY, hydroxylase CctR, and the bifunctional enzyme TruY, showed the diverse types of reactions catalyzed by BiNCOs using molecular oxygen as an oxidant and external ascorbate as reductant. Structurally, this family is defined by conserved dual HXXHC motifs that coordinate the binuclear copper center and an interchain disulfide‐linked homodimer structure. Furthermore, as BiNCOs are frequently associated with a specific family of fungal RiPPs, the study of AprY and TruY have shed light on the fungal RiPP maturation pathway. This review summarizes the current understanding of BiNCOs and provides an updated discussion of reaction mechanisms involved in halogenation, hydroxylation, macrocyclization, and aryl‐coupling reactions.