Identification of Amino Acid Residues Responsible for C−H Activation in Type‐III Copper Enzymes by Generating Tyrosinase Activity in a Catechol Oxidase
Ioannis Kampatsikas, Matthias Pretzler, Annette Rompel
Abstract
Abstract Tyrosinases (TYRs) catalyze the hydroxylation of phenols and the oxidation of the resulting o ‐diphenols to o ‐quinones, while catechol oxidases (COs) exhibit only the latter activity. Aurone synthase (AUS) is not able to react with classical tyrosinase substrates, such as tyramine and l ‐tyrosine, while it can hydroxylate its natural substrate isoliquiritigenin. The structural difference of TYRs, COs, and AUS at the heart of their divergent catalytic activities is still a puzzle. Therefore, a library of 39 mutants of AUS from Coreopsis grandiflora ( Cg AUS) was generated and the activity studies showed that the reactivity of the three conserved histidines (HisA 2 , HisB 1 , and HisB 2 ) is tuned by their adjacent residues (HisB 1 +1, HisB 2 +1, and waterkeeper residue) either to react as stronger bases or / and to stabilize a position permissive for substrate proton shuffling. This provides the understanding for C−H activation based on the type‐III copper center to be used in future biotechnological processes.