Laccase distribution

The laccase molecule is a dimeric or tetrameric glycoprotein, which contains four copper atoms per monomer, distributed in three redox sites. More than 100 types of laccase have been characterized. These enzymes are glycoproteins with molecular weights of 50-130 kDa. Approximately 45% of the molecular weight of this enzyme in plants are carbohydrate portions, whereas fungal laccases contain less of a carbohydrate portion (10-30%). Some studies have suggested that the carbohydrate portion of the molecule ensures the conformational stability of the globule and protects it from proteolysis and inactivation by radicals (Morozova and others 2007). 

Copper oxidases are widely distributed in nature, and enzymes from plants, microbes, and mammals have been characterized (104,105). The blue copper oxidases, which include laccases, ascorbate oxidases, and ceruloplasmin, are of particular interest in alkaloid transformations. The principle differences in specificity of these copper oxidases are due to the protein structures as well as to the distribution and environment of copper(II) ions within the enzymes (106). While an in vivo role in metabolism of alkaloids has not been established for these enzymes, copper oxidases have been used in vitro for various alkaloid transformations. 

Figure 14. Change (solid line) in apparent molecular weight distribution of water-soluble extract from a cellulase-treated ezomatsu wood residue (dotted line) at pH 4.0 brought about by the laccase HI preparation from C. versicolor, Sephadex GIO/H2O elution profiles adapted and redrawn from reference 85.

Laccase is perhaps the metallo-enzyme most widely used for this aim. Laccases are a family of multicopper ( blue copper ) oxidases widely distributed in nature Many laccases have fungal origin, while others are produced in plants. They contain four Cu(II) ions, and catalyse the one-electron oxidation of four molecules of a reducing substrate with the concomitant four-electron reduction of oxygen to water . In view of their low redox potential, which is in the range of 0.5-0.8 V vs. NHE depending on the fungal source laccases typically oxidize phenols (phenoloxidase activity) or anilines. 

Figure 3. Beech heartwood, decayed for 4 weeks with C. versicolor, showing thinning of the secondary wall with enlargement of the cavity at the cell corner (the protruding ends of the middle lamella can be seen). A single hypha close to the wood cell wall shows where the secondary wall has been degraded. Label for laccase is seen uniformly distributed in the secondary wall. Magnification x 12,600.

Figure 2.25. Transformation of catechol (0.1 M) in binary and ternary systems (A) Catechol removal by increasing concentrations of birnessite (B) catechol removal by increasing activities of Trametes villosa laccase (C) catechol removal by T. villosa laccase (950katalml 1) and birnessite (lmgml ) applied together (D) distribution of radioactivity after the incubation of 14C-labeled catechol with T. villosa laccase (950katalml ) and birnessite (lmgml-1).The reactions were carried out in 0.5% NaCl for 24h at 25°C. Reprinted from Ahn, M.-Y., Martinez, C. E., Archibald, D. D., Zimmerman, A. R., Bollag, J.-M., and Dec, J. (2006). Transformation of catechol in the presence of a laccase and birnessite. Soil Biol. Biochem. 38,1015-1020, with permission from Elsevier.

Claus, H. (2004). Laccases structure, reactions and distribution, Micron. 35, 93-96. 

Claus, H. 2004. Laccases Structure, reactions, distribution. Micron, 35 93-96. 

Laccase is widely distributed in plants and fungi. Laccase from higher plants, found in various species of the Chinese, Vietnamese, and Japanese lacquer trees, has been extensively investigated (9). The biological function of laccase in these trees is well understood. The laccase of the lacquer trees (Rhus sp.) is found in white latex, which contains phenols. After injury of the tree, these are oxidized by dioxygen to radicals, which spontaneously polymerize, building a protective structure that closes the wound. 

C (66). If electron transfer from type 1 to type 3 copper couples the two halves of the enzyme cycle, as proposed for laccase, then this intramolecular redox reaction must be extremely rapid to account for the effects of trace dioxygen on the reduction of the type 1 copper. Consequently, despite the fact that an ambiguous assignment of a type 1 to type 3 transfer is not possible in this example, facile intramolecular electron transfer processes probably ensure a rapid distribution of electrons among the type 1 and type 3 copper centers, at least in some of the enzyme molecules. The equilibrium distribution, and quite conceivably the relative rates of approach to this state, should be influenced by the oxidation-reduction potentials, which, as described earlier in this chapter (Figure 5), favor electron occupancy of the type 3 copper pairs at 10.0°C. 

An important family of multicopper enzymes couple the reduction of O2 to H2O with substrate oxidation. They include ascorbate oxidase, ceruloplasmin, Fet3, hephaestin, and laccase, and contain at least four copper ions. The four Cu ions are distributed between one type 1 blue copper site, one type 2 site, and one type 3 copper site. The blue Type 1 site is usually located some 12—13 A distant from a trinuclear site which has the two Type 3 coppers, linked by a bridging oxygen and one Type 2 copper. We illustrate this class of oxidases with laccase which catalyses the four-electron reduction of O2 to water, coupled with the oxidation of small organic... 

Laccases are so widely distributed in the fungi that it is very possible that this enzyme may be ubiquitous in these organisms [3,116], Laccases have also been found in a large variety of plant species [117], in about a dozen studied insects [118], and has also recently been found in the hacterium Azospirillum lipoferum [119]. Laccase has a variety of functions including participation in lignin biosynthesis [117], degradation of plant cell-walls [120,121], plant pathogenicity [122], and insect sclerotiza-tion [123]. 

Recently, laccases found some interest for synthetic application. Laccases are widely distributed in plants and fungi1131. The copper-containing enzymes are some of the few oxidases so far reported to reduce molecular oxygen to water (aside from cytochrome c oxidase and others). This ability was recently exploited in a novel regeneration concept for flavin-dependent enzymes (see Chapter 16.2)[14]. 

The distribution of these molecules is perfectly consistent with their antifungal properties, as they stop the mycelial development of fungi lacking in laccase, the only enzyme capable of breaking them down without being deactivated. The skin also contains phenolic acids and flavanols in the cell vacuoles. Phenolic acids are the main phenol components of the flesh. 

Holwerda and Gray (97) proposed a mechanism for the reduction process involving a central role of Type 2 Cu2+ as the initijil point at which electrons enter and are subsequently distributed to the other electron acceptors. This interpretation would seem to be supported by the very recent observation of Branden and Reinhammar (98) that the Type 2 ion of Poljqjorus laccase is reduced and subsequently reoxidized in a very short time period. The authors also emphasize the parallel behavior of the T q)e 1 and Type 3 centers it is particularly striking that under a variety of conditions the rates of Type 1 and 3 reductions are very similar. Indeed, when Cr2+ was used as the reductant, a similar observation was made (99). Perhaps Cr2+ reduces the enzyme via a bridging ligand (H2O ) between it and Type 2 Cu2+. 

The evaluation of tlie specific activity of laccase shows that it is not markedly diminished by the addition of the linear-dendritic copolymer (Table 2). It should be noted, however, that ABTS is a highly polar compound and its distribution balance between the dendritic domains on the laccase surface and the surrounding water will be strongly shifted towards the aqueous phase ultimately affecting the specific activity of the complexes. Therefore the more hydrophobic HBT (Figure 3B) is chosen as the mediator for the next model... 

Rhus vernicifera laccase is known to incorporate four tightly bound copper atoms distributed in three distinct sites. Type 1 copper is responsible for the intense blue colouration, and a second form of metal ion, type 2, is not associated with any specific spectroscopic absorption bands but has been inferred from e.s.r. studies. This centre is known to function as a strong anion-binding site. Type 3 copper is non-detectable by e.s.r. and is characterized by a u.v. absorption band (Amax ca. 330 nm). Anaerobic stopped-flow studies with hydroquinone (HgQ) have been made to investigate the mode of reduction at these centres. The type 1 and type 3 copper ions are reduced in parallel at comparable rates over a wide range of substrate concentrations and pH. The rate data are consistent with the mechanism... 

AOx, CP, and tree and fungal laccases (Figure 8.5) [69]. Comparisons among the ground-state distribution of the Cu—S(cys) stretching vibration in the T1 copper site in each of these proteins show that they differ in strength. The Cu—S(cys) bond is one of the three essential coordination complexes of copper that defines the T1 site in MCOs and other copper proteins and is the major factor in electron transfer from the substrate to the trinuclear center [40,85]. Hence, RR spectroscopy can provide valuable information on how subatomic structural variations at this coordination can drive the functional properties of electron transfer at the T1 site. 

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