Cyclochlorotine Hydroxylase CctR Reveals DUF3328 as a Family of Copper‐Dependent Metalloenzymes

DUF3328 is a protein family widely found in fungal natural product biosynthesis pathways. Although DUF3328 proteins have long been implicated in diverse modifications of inert C( sp 3 )─H bonds, including halogenation, hydroxylation, and macrocyclization, the biochemical properties and catalytic mechanisms of DUF3328 proteins remain elusive. Here, we report the characterization of the DUF3328 protein CctR, which catalyzes C( sp 3 )─H hydroxylation of fungal cyclic peptide cyclochlorotine. Through AlphaFold modeling, in vitro biochemical characterization, and spectroscopic analysis, we demonstrate that CctR is a membrane‐associated copper‐dependent enzyme that functions as a homodimer. The dimerization of CctR is mediated by its transmembrane helix, a four‐helix coiled coil, and C‐terminal disulfide bonds. The conserved HxxHC(x) n HxxHC motif, characteristic of the DUF3328 superfamily, is anchored on the dimerization interface and forms a binuclear copper coordination center. Moreover, we show that CctR is dioxygen‐dependent and requires electron input for the hydroxylation reaction. Together, these findings define DUF3328 as a previously unrecognized family of binuclear copper‐dependent metalloenzymes, capable of catalyzing diverse chemical transformations, and lay the groundwork for future discovery of novel biocatalysts within this widespread enzyme class.