Identification of Amino Acid Residues Responsible for C−H Activation in Type‐III Copper Enzymes by Generating Tyrosinase Activity in a Catechol Oxidase

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 (CgAUS) was generated and the activity studies showed that the reactivity of the three conserved histidines (HisA2, HisB1, and HisB2) is tuned by their adjacent residues (HisB1+1, HisB2+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.

Similar but Still Different: Which Amino Acid Residues Are Responsible for Varying Activities in Type‐III Copper Enzymes?

“…E) ortho ‐hydroxylation of the phenolate by an electrophilic aromatic substitution and the subsequent two‐electron oxidation of the diphenolic intermediate yield the final ortho ‐quinone product, and one molecule of water. During the two‐electron oxidation step, the PPO copper center is reduced to its deoxy‐form, closing the catalytic monophenolase cycle [45] . Diphenolase activity (red): C) The diphenolic substrate is oxidized to the corresponding quinone by the dicopper center, which transitions from the oxy‐ to the met‐form.…”

“…In all the mutants in which the Phe gatekeeper residue was changed to a smaller amino acid, the enzyme‘s original activity was impaired. However, the Phe273Leu mutant of Cg AUS produced monophenolase activity with the classical monophenolic substrates ( l ‐tyrosine and tyramine), indicating that the gatekeeper residue (Phe) does impede the tyrosinase activity [45] . The gatekeeper residue therefore has a dual function and either supports (π‐π interactions with Phe or free entry for the substrate with a small amino acid at this position) or inhibits (blocks the access) the substrate entry in plant PPOs.…”

“…Thus, they assist HisB 1 in finding the best position for activation (Asp) so that HisB 1 can react as a base to deprotonate the incoming monophenol, thereby initiating the hydroxylation reaction (Figure 2). [45] The designation first activity controller truly characterizes the position HisB 1 +1 in the type‐III copper enzymes. The majority of the mutants have an pronounced effect on the monophenolase and diphenolase activity (Table 4).…”

Considerations on activity determinants of fungal polyphenol oxidases based on mutational and structural studies

“…On the other hand, the side chain carbonyl of Asn292 is at 3.7 Å distance from nitrogen ND1 of H B1 (His291) ( Fig. 6A) and could thus form a hydrogen bond with the latter through a minimal rearrangement upon substrate binding, rendering H B1 a potential base for the deprotonation of incoming monophenols as recently suggested (19). This is however not in accordance with the biochemical data, which did not show any increase of the monophenolase activity of this variant.…”

“…Crystallographic work on A. oryzae TYR also pointed out the migration of both copper ions and the detachment of H A3 , potentially acting as a base to accept a proton from the bound tyrosine substrate (18). The increased flexibility of copper coordinating His residues (H A2 , H B1 and H B2 ) and their role as deprotonating bases was further corroborated by measuring the increase in monophenolase activity of various of CgAUS mutants (19).…”