Axial bonds at the T1 Cu site of Thermus thermophilus SG0.5JP17‐16 laccase influence enzymatic properties

Abstract

Laccase is a multi‐copper oxidase which oxidizes substrate at the type 1 copper site, simultaneously coupling the reduction of dioxygen to water at the trinuclear copper center. In this study, we used site‐directed mutagenesis to study the effect of axial bonds between the metal and amino acid residue side chains in lac TT . Our kinetic and spectral data showed that the replacement of the axial residue with non‐coordinating residues resulted in higher efficiency ( k cat / K m ) and a lower Cu 2+ population at the type 1 copper site, while substitution with strongly coordinating residues resulted in lower efficiency and a higher Cu 2+ population, as compared with the wild‐type. The redox potentials of mutants with hydrophobic axial residues (Ala and Phe) were higher than that of the wild‐type. In conclusion, these insights into the catalytic mechanism of laccase may be of use in protein engineering to fine‐tune its enzymatic properties for industrial application.

Mutations in the coordination spheres of T1 Cu affect Cu2+-activation of the laccase from Thermus thermophilus

“…For M455L Tt-LAC, the onset for O2 reduction is shifted anodic to 650 mV vs NHE. This shift of almost 100 mV confirms the reported influence of leucine ligand against methionine to promote high T1 Cu redox potentials in various MCOs [31]. It was proposed that this anodic shift would be linked to the stabilization of the reduced state of Cu in the absence of methionine axial ligand.…”

“…Absorbance peak decrease at 607 nm suggests some depletion of T1 Cu site in M455L mutant. It is noticeable however, that our protocol for copper incorporation is much more efficient than the protocol recently used by Zhu et al [31]. T2/T3 active site is attested by a weakdefined band at 330 nm for Tt LAC WT, induced by a bridging hydroxo ligand between T3 coppers.…”

“…In particular, M455, the axial ligand of T1 Cu, was replaced by leucine (L) and phenylalanine (F), as this mutation was previously shown in classical MCOs to induce an increase in the T1 Cu redox potential [30,31]. M456 was mutated to alanine (A) recognized as a conserved residue in high potential LACs.…”

Electrochemistry of copper efflux oxidase-like multicopper oxidases involved in copper homeostasis

“…Site directed mutagenesis was realized in Tt LAC and McoP by substitution of the Met CuT1 axial ligand to Leu or Phe. Direct electrochemistry experiments underlined a positive shift between 60 and 100 mV of the onset potential for O2 reduction [22,57,58]. Poor stability of the mutants however precluded an enhancement of the activity.…”

Laccase-Based Biosensor Encapsulated in a Galactomannan-Chitosan Composite for the Evaluation of Phenolic Compounds

“…This point shows the interest of using a galactomannan = chitosan composite rather than pure galactomannan for the electrochemical detection. After encapsulation of laccase, the peak maximum intensities are doubled, due to the presence of copper in the active site of the enzyme [24].…”