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

Abstract

Type‐III copper enzymes like polyphenol oxidases (PPOs) are ubiquitous among organisms and play a significant role in the formation of pigments. PPOs comprise different enzyme groups, including tyrosinases (TYRs) and catechol oxidases (COs). TYRs catalyze the o‐hydroxylation of monophenols and the oxidation of o‐diphenols to the corresponding o‐quinones (EC 1.14.18.1). In contrast, COs only catalyze the oxidation of o‐diphenols to the corresponding o‐quinones (EC 1.10.3.1). To date (August 2020), 102 PDB entries encompassing 18 different proteins from 16 organisms and several mutants have been reported, identifying key residues for tyrosinase activity. The structural similarity between TYRs and COs, especially within and around the active center, complicates the elucidation of their modes of action on a structural basis. However, mutagenesis studies illuminate residues that influence the two activities and show that crystallography on its own cannot elucidate the enzymatic activity mode. Several amino acid residues around the dicopper active center have been proposed to play an essential role in the two different activities. Herein, we critically review the role of all residues identified so far that putatively affect the two activities of PPOs.

Expression, Purification, and Characterization of a Well-Adapted Tyrosinase from Peatlands Identified by Partial Community Analysis

“…Enzymatic activity, substrate preferences, and pH dependency of TYRs are controlled by a framework of second shell amino acids located in and around the catalytic pocket. ,,,, In bacterial TYRs, mono- and diphenolase activities have been observed for all investigated enzymes so far; however, the first (His B1 + 1) and second (His B2 + 1) activity controllers critically influence the kinetic behavior and substrate acceptance. ,,,, The high level of heterogeneity in both the overall TYR sequences and the amino acids featured in the position of the activity controller residues suggests that TYR enzymes present in peatlands act on a broad scope of substrates and, therefore, have the potential of efficiently removing phenolic compounds with increased aeration of previously anoxic peat layers. In addition, the degenerated type III copper protein primers designed within this study represent a valuable tool for further partial community analysis of TYR-producing organism via , e.g ., single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE).…”

Evidence for H-bonding interactions to the μ-η2:η2-peroxide of oxy-tyrosinase that activate its coupled binuclear copper site

“…However, putative differences in the electronic/geometric structures of their Cu(II) 2 O 2 active sites as well as the molecular-level factors that control their reactivity are not fully understood despite extensive research efforts. 8,[14][15][16] Second-sphere interactions involving active-site residues and/or solvent molecules with the Cu(II) 2 O 2 core could have important mechanistic implications, but have been challenging to define experimentally, in part, due to the fact that most of the structural insights for catalytic oxy-CBC enzymes have derived from crystallographic studies on their latent pro-forms (PDB : 6JUA-D) 17 or binary complexes with their caddie proteins (PDB : 1WX2, 1WX4-5), 4 where in all cases the Ty active sites are occupied by additional residues, paralleling oxy-Hc (Fig. S4, ESI †).…”

“…2C), however the functional significance of these second-sphere correlations remains a subject of debate. 14,16,21,[25][26][27] Inspection of our experimentally-calibrated structure of the oxy-Ty active site reveals that these key second-sphere residues are critical in allowing solvent access to the active site and stabilizing W2, and thus enable the H-bonding to the m-Z 2 :Z 2 -peroxide (Fig. 2B).…”

Considerations about the Continuous Assay Methods, Spectrophotometric and Spectrofluorometric, of the Monophenolase Activity of Tyrosinase

“…This difference in activities of the Em and Eox forms is the origin of the kinetic complexity of the tyrosinase monophenolase activity. The EmM complex is inactive and therefore for the enzyme to be active on monophenols it is necessary for the Em form to be reduced to originate the Ed form and its binding to the oxygen to form Eox, closing the catalytic cycle (Scheme 1) [5][6][7]. There is no such problem in diphenolase activity as the two forms Em and Eox are active on o-diphenol (D) (Scheme 2) [5].…”

“…However, with respect to monophenols, the Em form is inactive, forming an EmM dead-path complex. The Eox form is active on monophenols, carrying out its hydroxylation to o -diphenols through an electrophilic aromatic substitution [ 5 , 6 , 7 ]. This difference in activities of the Em and Eox forms is the origin of the kinetic complexity of the tyrosinase monophenolase activity.…”