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

Abstract

Thermus thermophilus laccase belongs to the sub-class of multicopper oxidases that is activated by the extra binding of copper to a methionine-rich domain allowing an electron pathway from the substrate to the conventional first electron acceptor, the T1 Cu. In this work, two key amino acid residues in the 1 st and 2 nd coordination spheres of T1 Cu are mutated in view of tuning their redox potential and investigating their influence on copper-related activity. Evolution of the kinetic parameters after copper addition highlights that both mutations play a key role influencing the enzymatic activity in distinct unexpected ways. These results clearly indicate that the methionine rich domain is not the only actor in the cuprous oxidase activity of CueO-like enzymes.

Nanomaterials as Redox Mediators in Laccase-Based Amperometric Biosensors for Catechol Assay

“…The traditional understanding of the mechanisms of laccase catalytic activity was that laccase does not require H 2 O 2 as a co-substrate or an additional cofactor for an enzymatic reaction, nor does laccase produce this compound [ 51 ]. However, that postulate was disproven in past decades, when an alternative mechanism of O 2 reduction mediated with laccase was discovered [ 1 , 2 , 7 , 8 , 52 , 53 , 54 , 55 , 56 , 57 , 58 ].…”

“…A number of recombinant and mutant strains have been proposed [ 1 , 2 , 3 , 4 , 5 , 9 , 14 ] to improve the properties of this enzyme which plays important roles in many fields of science, biotechnology, industry, and medicine. Various properties of LMCO were studied, including substrate specificity, structure, and different mechanisms of catalytic activity [ 1 , 2 , 3 , 7 , 52 , 53 , 54 , 55 , 56 , 57 , 58 ].…”

“…The electron transfer (ET) process in laccases, and the mechanism of O 2 reduction as elucidated through spectroscopic, kinetic, crystallographic, and computational data, have been described in several papers [ 1 , 2 , 3 , 52 , 53 , 54 , 55 , 56 ]. Laccases classed as LMCOs contain a minimum of four copper sites, designated as T1, T2, and binuclear T3.…”

“…The main approaches to investigating the ET mechanism are spectroscopic characterization of reaction intermediates, kinetic analysis, oxidation of substrates designed to serve as mechanistic probes, the effect of the addition of radical traps, X-ray devices, and computational analysis [ 2 , 3 , 7 , 53 , 54 , 55 , 56 , 57 , 58 ]. As the result of these high-throughput physical methods, formation of two sequential intermediates, namely “peroxide (PI)” and the “native (NI)”, were hypothesized.…”

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

“…The search for a good balance between stability and activity of enzymes is a prerequisite, even complexified by the search for efficient ET in the case of redox enzymes. Recent examples in the literature concerning MCOs revealed the difficulty of getting improved interfacial ET parameters or improved redox properties while maintaining both the stability and the activity of the enzyme [300,301]. No doubt that protein engineering, which is increasingly carried out with the aid of innovative solutions based on emerging genetic tools, will offer new opportunities of producing stable enzymes.…”