Crystal structure of a blue laccase from Lentinus tigrinus: evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases

Marta Ferraroni,Fabrizio Briganti,Andrea Scozzafava,Alexey A Leontievsky,Nina M Myasoedova,Vadim Schmatchenko,Ludmila A Golovleva

doi:10.1186/1472-6807-7-60

Abstract

BackgroundLaccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O2 reduction to H2O.ResultsThe structure of a laccase from the white-rot fungus Lentinus (Panus) tigrinus, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 Å. It reveals a asymmetric unit containing two laccase molecules (A and B). The progressive reduction of the copper ions centers obtained by the long-term exposure of the crystals to the high-intensity X-ray synchrotron beam radiation under aerobic conditions and high pH allowed us to detect two sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermediates, previously hypothesized through spectroscopic, kinetic and molecular mechanics studies. Specifically the electron-density maps revealed the presence of an end-on bridging, μ-η1:η1 peroxide ion between the two T3 coppers in molecule B, result of a two-electrons reduction, whereas in molecule A an oxo ion bridging the three coppers of the T2/T3 cluster (μ3-oxo bridge) together with an hydroxide ion externally bridging the two T3 copper ions, products of the four-electrons reduction of molecular oxygen, were best modelled.ConclusionThis is the first structure of a multicopper oxidase which allowed the detection of two intermediates in the molecular oxygen reduction and splitting. The observed features allow to positively substantiate an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing general insights into the reductive cleavage of the O-O bonds, a leading problem in many areas of biology.

Highlights

Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances
Primary sequence The nucleotide sequence of L. tigrinus laccase gene was obtained as reported in the experimental procedures, the deduced sequence resulted to be composed by 463 amino acids
The sequence of L. tigrinus blue laccase gene lacks some 3' end nucleotides as in the gene amplifying we have used the Overall structure The crystal structure of LtL was solved at 1.5 Å resolution by using the molecular replacement technique and employing, as a starting model, the coordinates of the Coprinus cinereus laccase

Summary

Introduction

Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. The simplest reactions catalyzed by laccases are those in which a vast set of substrates, typically phenols and arylamino compounds, is oxidized to the corresponding radical species by direct interaction with their active site [3] and accompanied by the reduction of molecular oxygen to water Due to their very broad substrate range they are implicated in a extensive series of functions such as pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances such as lignin, humic matter, and toxic xenobiotics [4]. These non enzymatic routes of oxidative polymerizing or depolymerizing reactions are vital in a range of physiological functions such as lignolysis, lignin synthesis, morphogenesis, pathogenesis and detoxification

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