Intramolecular Coupling and Nucleobase Transfer – How Cytochrome P450 Enzymes GymBx Establish Their Chemoselectivity

Cyclodipeptides (CDPs) build an important class of secondary metabolites with a plethora of pharmacological impact such as antibiotic, cytotoxic, antifungal properties or even as anti‐Alzheimer drug candidates. The core structure of 2,5‐diketopiperazines, a subclass of CDPs, is composed of two ‘head‐to‐tail’ connected α‐amino acids. Their chemical diversity is broadened by tailoring enzymes as members of their biosynthetic gene clusters, such as CDP‐dimerizing P450s. Recently, a new class of P450 enzymes, termed GymB x , was identified in the biosynthetic pathway of guatyromycines. These enzymes catalyse both an intramolecular C−C bond formation within cyclo ‐ l ‐Tyr‐ l ‐Tyr (cYY) and a nucleobase transfer reaction, which is unique for CDP‐modifying P450 enzymes. In this study, we report the structures of two members, GymB 1 and GymB 5 , in their unbound states, as well as GymB 5 in complex with hypoxanthine and cYY. Structural data of the ternary complex shed light into CDP binding and nucleobase transfer reaction, identified key residues for substrate recognition and modified the enzymatic bifunctionality. By mutagenesis we successfully established a GymB5 variant that strictly switched the chemoselectivity of the reaction towards an intermolecular coupling enzyme. This data set a solid basis for future protein engineering of P450 enzymes to synthesize new CDP‐nucleobase adducts.