Laccases dimeric

Gierer, J. Opara, A. E. Enzymic degradation of lignin. Action of peroxidase and laccase on monomeric and dimeric model compounds. Acta Chem. Scand. 1973, 27,... 

Many multiple copper containing proteins (e.g., laccase, ascorbate oxidase, hemo-cyanin, tyrosinase) contain so-called type III copper centers, which is a historical name (cf. Section 5.8 for type I and type II copper) for strongly exchange-coupled Cu(II) dimers. In sharp contrast to the ease with which 5=1 spectra from copper acetate are obtained, half a century of EPR studies on biological type III copper has not produced a single triplet spectrum. Why all type III centers have thus far remained EPR silent is not understood. 

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). 

Dimeric aldehydes analogous to (II) and (VI) have in fact been isolated from incubates of coniferyl alcohol with laccase (58). There are only 3% such aldehydic groups in lignin 4) [cf. Unit 10 in Fig. 9] but these suffice to give a intense red color with phloroglucinol and concentrated hydrochloric acid, the conventional Wiesner test for lignin. 

The degradation products of GOG were (Vm), GC -Dimer, vanillin (II), and dehydrodivanillin (X). The reaction of GOG by laccase III, therefore, brings about the formation of biphenyl structures, the cleavage of C-C bond between a- and -carbons, and the cleavage of -0-4 ether linkages. The mode of these cleavages is similar to that of SOG. 

Hie GOG-Dimer formed from the lignin dimer compound (GOG) by laccase III was also degraded by laccase HI, resulting in the formation of compounds (I), (VI), and biphenyl dimer of 2-methoxy-l,4-benzoquinone (DC). This indicates that GOG-Dimer also suffers the cleavage of -0-4 ether linkage via the alkyl-phenyl cleavage. 

Figure 3.2 Coupled reaction of tyrosinase and laccase in conversion of tyrosol to dimeric products [48].

A possible reductive role for veratryl alcohol oxidase is proposed in Figure 5. Laccases from C. versicolor can produce both polymerization and depolymerization of lignin (29). In phenolic lignin model dimers, laccase can perform the same electron abstraction and subsequent bond cleavage as found for lignin peroxidase (30). The phenolic radical is however likely to polymerize unless the quinoid-type intermediates can be removed, for example by reduction back to the phenol. Veratryl alcohol oxidase, in... 

Each of these proteins is blue and appears to have a minimum of four copper atoms per molecule one type 1, one type 11, and two type III. Laccase is not known to be multimeric, nor is ceruloplasmin, but ascorbate oxidase is apparently a dimer. 

The blue oxidases contain these three types of copper together The stoichiometry is straightforward with laccase which contains one type-1 and one type-2 copper, and one type-3 dimeric copper site . One would expect two laccase-like sites in ascorbate oxidase and in ceruloplasmin, but the presence of respectively 3 and 1 and 1 and 3 type-1 and type-2 copper atoms has been deduced. Ceruloplasmin shows oxidase activities towards different substrates, like Fe (ferroxidase) and aromatic amines. It plays, moreover, an active role in the transport of copper With the proper precautions against the action of proteinases it can be isolated as a single polypeptide chain... 

Copper-catalyzed oxidations of phenols by dioxygen have attracted considerable interest owing to their relevance to enzymic tyrosinases (which transform phenols into o-quinones equation 24) and laccases (which dimerize or polymerize diphenols),67 and owing to their importance for the synthesis of specialty polymers [poly(phenylene oxides)]599 and fine chemicals (p-benzoquinones, muconic acid). A wide variety of oxidative transformations of phenols can be accomplished in the presence of copper complexes, depending on the reaction conditions, the phenol substituents and the copper catalyst.56... 

Type III copper(II), found for example in Rhus laccase, is ESR inactive. Although copper(II) is present no ESR spectrum can be obtained. Recent magnetic susceptibility measurements on Rhus laccase indicate an antiferromagnetically coupled cop-per(II) dimer. 

Cichewicz RH, Clifford LJ, Lassen PR, Cao X, Freedman TB, Nafie LA, Deschamps JD, Kenyon VA, Flanary JR, Holman TR, Crews P (2005) Stereochemical determination and bioactivity assessment of (S)-(l)-curcuphenol dimers isolated from the marine sponge Didiscus aceratus and synthesized through laccase biocatalysis. Bioorg Med Chem 13 ... 

Niemetz, R. and Gross, G.G. (2003b) Ellagitannin biosynthesis laccase-catalyzed dimerization of tellimagrandin II to cornusiin E in Tellima grandiflora. Phytochemistry, 64,1197-1201. 

Some efforts applying biotransformation, e.g. horseradish peroxidase (HRP), laccase-like stilbene-oxidase and Botrytis cinerea have led to some other new dimers resveratrol trans-dehydrodimer (553) [249-251], resveratrol cw-dehydrodimer (556) [249,251], pterostilbene... 

The microbial transformations of vindoline and its derivatives have been further examined. Human caeruloplasmin and laccases of Polyporus anceps and Rhus vernicifera converted vindoline (44) into the known enamine 270 and the dimer 266 (201), which have been encountered in previous microbiological studies on vindoline. The dimer 266 is also formed by the action of horseradish peroxidase on vindoline (202). 

To better understand the mechanisms for the oxidation of lignin by a laccase mediator system, a laccase from Polyporous sp, kindly provided by Novozymes, was used in combination with 1-HBT. The redox mediator was found to be partly regenerated during the oxidation of lignin dimer 1 in the presence of laccase. A free radical of 1-HBT generated by laccase was probably responsible for the oxidation of I [146]. The free radical of 1-HBT was, however, transformed to benzotriazole, which could not mediate the oxidation of I. A proposed mechanism for the laccase mediator oxidation of nonphenolic lignins is given in Scheme 14.1. 

To investigate the importance, not only of laccase mediators, but also of lacca-ses per se, several laccases were studied for the oxidation of the nonphenolic lignin dimer I. In the presence of the redox mediators 1-HBT or violuric acid, it was found that the oxidation rates of dimer I by the laccases differed considerably. In oxidation of dimer I, both 1-HBT and violuric acid were to some extent, consumed. The consumption rate followed the same order of laccases as the oxidation rates of dimer I. The oxidation rate of dimer I was found to be dependent on both k, jt and the stability of the laccase in question. Both 1-HBT and violuric acid inactivated the laccases— violuric acid to a greater extent then 1-HBT. The presence of dimer I in the reaction mixture slowed down this inactivation. Inactivation seems to be mainly due to the reaction of the redox mediator free-radical with the laccases. No relationship between the carbohydrate content of the laccases and their inactivation was found. When the redox potential of the laccases is in the range of 750-800 mV, i.e., about that of the redox mediator, a further increase in redox potential does not affect k(,jt and the oxidation rate of dimer I [147]. 

FIGURE 15.12 Lignin model dimers oxidation catalyzed by laccase and HBT. (From Bourbonnais, R., Paice, M.G., Freiermuth, B., Bodie, E., Borneman, S., Applied and Environmental Microbiology, 63(12), 4627-4632, 1997. With permission.)... 

S Kawai, M Nakagawa, H Ohashi. Aromatic ring cleavage of a non-phenohc P-OA lignin model dimer by laccase of Trametes versicolor in the presence of 1-hydroxyben-zotriazole. F S5 Leff. 446(2-3) 355-358, 1999. 

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