Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one

Abstract

Tyrosinase(EC 1.14.18.1) catalyzes the monophenolase and diphenolase reaction associated with vertebrate pigmentation and fruit/vegetable browning. Tyrosinase is an oxygen-dependent, dicopper enzyme that has three states: Emet, Eoxy, and Edeoxy. The diphenolase activity can be carried out by both the met and the oxy states of the enzyme while neither mono- nor diphenolase activity results from the deoxy state. In this study, the oxidative cyclocondensation of 2-aminophenol (OAP) to the corresponding 2-aminophenoxazin-3-one (APX) by mushroom tyrosinase was investigated. Using a combination of various steady- and pre-steady state methodologies, we have investigated the kinetic and chemical mechanism of this reaction. The kcat for OAP is 75±2 sec−1, KMOAP=1.8±0.20.2emnormalmM, KMO2=25±40.2emnormalμitalicM with substrates binding in a steady-state preferred fashion. Stopped flow and global analysis support a model where OAP preferentially binds to the oxy form over the met (k7 ≫ k1). For the met form, His269 and His61 are the proposed bases, while the oxy form uses the copper-peroxide and His61 for the sequential deprotonation of anilinic and phenolic hydrogens. Solvent KIEs show proton transfer to be increasingly rate limiting for kcat/KMOAP as [O2] −>0 μM (1.38±0.06) decreasing to 0.83±0.03 as [O2]−>∞ reflecting a partially rate limiting μ-OH bond cleavage (Emet) and formation (Eoxy) following protonation in the transition state. The coupling and cyclization reactions of o-quinone imine and OAP pass through a phenyliminocyclohexadione intermediate to APX, forming at a rate of 6.91±0.03 μM−1s−1 and 2.59E-2±5.31E-4 s−1. Differences in reactivity attributed to the anilinic moiety of OAP with o-diphenols are discussed.

A missense mutation in TYRP1 causes the chocolate plumage color in chicken and alters melanosome structure

“…Each metal‐ion‐binding site (A and B) contains three conserved histidine residues (Furumura et al, ; Lai et al, ). Together, they bind directly to the metal ions in mammalian TYRP1 (García‐Borrón & Solano, ) and fungal (Washington et al, ) TYR. The missense mutation (His214Asn) detected in our study disrupts one of those histidine residues interacting with a metal ion and is therefore expected to have a large negative effect on TYRP1 function.…”

Catalysis-based specific detection and inhibition of tyrosinase and their application

“…There is a large difference between monophenolase activity and diphenolase activity, and reduced tyrosinase obviously has a lag phase when reacting with monophenol. These are important topics that need to be continuously explored and studied [ 11 ].…”

“…In addition, it plays an indispensable role in killing parasitic plants against phenolic symbiotic bacteria, the production of natural pigments and the synthesis of amino acid antibiotics such as lincomycin. The common point of these effects is inseparable from the tyrosinase using redox reactions with oxygen molecules [ 11 ].…”

Questiomycins, Algicidal Compounds Produced by the Marine Bacterium Alteromonas sp. D and Their Production Cue

“…It is reported that 2-aminophenoxazin-3-one (APO) core structure of bacterial products can be formed by oxidative coupling of two o-aminophenols catalyzed by various types of oxidases [32][33][34]. We therefore supplemented o-aminophenol to the Alteromonas sp.…”