Broad substrate scope C-C oxidation in cyclodipeptides catalysed by a flavin-dependent filament

Cyclic dipeptides are produced by organisms in all domains of life. Many possess biological activities as anticancer and antimicrobial compounds. Oxidations are frequently present in these biologically active peptides. C-C bond oxidation is catalysed by tailoring enzymes, including cyclodipeptide oxidases. These flavin-dependent enzymes are underexplored due to their complex three-dimensional arrangement involving multiple copies of two distinct small subunits and unclear mechanistic features underlying substrate selection and catalysis. Here, we determined the structure and mechanism of the cyclodipeptide oxidase from the halophile Nocardiopsis dassonvillei ( Ndas CDO), part of the biosynthetic pathway for nocazine natural products. We show Ndas CDO forms filaments in solution with a covalently bound FMN cofactor in the interface between three distinct subunits. The enzyme is promiscuous, using many cyclic dipeptides as substrates in a distributive manner. The reaction is optimal at high pH, involving the formation of a radical intermediate. Pre-steady state kinetics, a sizeable solvent kinetic isotope effect and lack of viscosity effects support that a step coupled to FMN regeneration determines the rate of the reaction. Our work dissects the complex mechanistic and structural features of this dehydrogenation reaction. This sets the stage to utilizing Ndas CDO as a biocatalyst and expands the FMN-dependent oxidase family to include enzyme filaments. ### Competing Interest Statement The authors have declared no competing interest. * EPR : Electron paramagnetic resonance LC-MS : liquid chromatography mass spectrometry Ndas CDO : cyclodipeptide oxidase from Nocardiopsis dassonvileii CDPS : cyclodipeptide synthase CDP : cyclodipeptide. All cyclic dipeptides used here were composed by L-amino acids and abbreviated with the letter “c” for cyclo, followed by the 1 letter code for each amino acid as follows: cFG, cFP, cFF, cLP, cHF, cWG, cWW, cWY, cWS