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

Department of Chemistry University of Florence, Via della Lastruccia 3, Sesto Fiorentino, 50019 Florence, Italy.
BMC Structural Biology (Impact Factor: 2.22). 02/2007; 7(1):60. DOI: 10.1186/1472-6807-7-60
Source: PubMed

ABSTRACT 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. 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.
The structure of a laccase from the white-rot fungus Lentinus (Panus) tigrinus, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 A. 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, micro-eta 1:eta 1 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 (micro3-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.
This 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.

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Available from: Ludmila A Golovleva, Aug 26, 2015
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    • "In order to decipher the oxidation mechanism of laccases, it is critical to understand its three dimensional structure and amino acid (AA) residue sequence. Several studies on laccase from different species of fungi have recently provided a significant insight of its crystalline structure and AA residues sequence (Ducros et al., 1998; Ferraroni et al., 2007; Hakulinen et al., 2002; Piontek et al., 2002; Garavaglia et al., 2004; Kallio et al., 2011; Bertrand et al., 2002) that is summarized hereafter. "
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    ABSTRACT: Over the last few decades many attempts have been made to use biocatalysts for the biotransformation of emerging contaminants in environmental matrices. Laccase, a multicopper oxidoreductase enzyme, has shown great potential in oxidizing a large number of phenolic and non-phenolic emerging contaminants. However, laccases and more broadly enzymes in their free form are biocatalysts whose applications in solution have many drawbacks rendering them currently unsuitable for large scale use. To circumvent these limitations, the enzyme can be immobilized onto carriers or entrapped within capsules; these two immobilization techniques have the disadvantage of generating a large mass of non-catalytic product. Insolubilization of the free enzymes as cross-linked enzymes (CLEAs) is found to yield a greater volume ratio of biocatalyst while improving the characteristics of the biocatalyst. Ultimately, novel techniques of enzymes insolubilization and stabilization are feasible with the combination of cross-linked enzyme aggregates (combi-CLEAs) and enzyme polymer engineered structures (EPESs) for the elimination of emerging micropollutants in wastewater. In this review, fundamental features of laccases are provided in order to elucidate their catalytic mechanism, followed by different chemical aspects of the immobilization and insolubilization techniques applicable to laccases. Finally, kinetic and reactor design effects for enzymes in relation with the potential applications of laccases as combi-CLEAs and EPESs for the biotransformation of micropollutants in wastewater treatment are discussed.
    Critical Reviews in Biotechnology 10/2012; 33(4). DOI:10.3109/07388551.2012.725390 · 7.84 Impact Factor
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    • "r - vation carried out on high E 0 ( >700 mV ) Lentinus tigrinus laccase ( LtL - 2QT6 ) confirmed the presence of five phenylalanine residues in the near surrounding of T1 and of two further hydrophobic residues ( Ile425 and Phe462 ) at about 3 . 5 – 3 . 6 Å , which were altogether suggested to contribute to the high E 0 observed for this enzyme ( Ferraroni et al . , 2007 ) . Furthermore , the occurrence of a large number of hydrophobic residues ( Phe329 , Phe336 , Ile338 , Ala386 , Phe396 , and Ile452 ) belonging to the four loops delineating the substrate cavity of the high E 0 laccase ( 760 mV ) from Trametes trogii ( TtL - 2HRG ) had been reported ( Matera et al . , 2008 ) . Consistently , we have id"
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    ABSTRACT: Laccases are multicopper oxidases in which substrate oxidation takes place at the type-1 (T1) copper site. The redox potential (E (0)) significantly varies amongst members of the family and is a key parameter for substrate specificity. Despite sharing highly conserved features at the T1 copper site, laccases span a large range of E (0), suggesting that the influence of the metal secondary coordination sphere is important. In silico analysis of structural determinants modulating the E (0) of Rigidoporus lignosus and other fungal laccases indicated that different factors can be considered. First, the length of the T1 copper coordinating histidine bond is observed to be longer in high E (0) laccases than in low E (0) ones. The hydrophobic environment around the T1 copper site appeared as another important structural determinant in modulating the E (0), with a stronger hydrophobic environment correlating with higher E (0). The analysis of hydrogen bonding network (HBN) around the T1-binding pocket revealed that the amino acids building up the metal binding site strongly interact with neighbouring residues and contribute to the stabilization of the protein folds. Changes in these HBNs that modified the Cu1 preferred coordination geometry lead to an increase of E (0). The presence of axial ligands modulates the E (0) of T1 to different extent. Stacking interactions between aromatic residues located in the second coordination shell and the metal ion coordination histidine imidazole ring have also been identified as a factor that modulates the E (0). The electrostatic interactions between the T1 copper site and backbone carbonyl oxygen indicate that Cu1-CO=NH distance is longer in the high E (0) laccases. In short, the in silico study reported herein identifies several structural factors that may influence the E (0) of the examined laccases. Some of these are dependent on the nature of the coordination ligands at the T1 site, but others can be ascribed to the hydrophobic effects, HBNs, axial ligations, stacking and electrostatic interactions, not necessary directly interacting with the copper metal.
    Journal of biomolecular Structure & Dynamics 02/2012; 30(1):89-101. DOI:10.1080/07391102.2012.674275 · 2.98 Impact Factor
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    • "Most laccases consist of three cupredoxin-like domains and contain four copper ions that reside in a T1 copper site and a T2/T3 tricopper center (Zhukhlistova et al., 2008). The copper ions are coordinated by highly conserved residues in domains I and III, whereas the substrate binding pocket is formed from less conserved residues in domains II and III (Bertrand et al., 2002; Ferraroni et al., 2007; Kallio et al., 2011, 2009; Matera, 2008). A substrate binds near the T1 copper site and is oxidized by the transfer of an electron to the T1 copper (Zhukhlistova et al., 2008). "
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    Insect biochemistry and molecular biology 12/2011; 42(3):193-202. DOI:10.1016/j.ibmb.2011.11.010 · 3.42 Impact Factor
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