Laccases pathways

The monophenolase activity of PPO is generally defined as the first step in the melaniza-tion pathway and consists of the o-hydroxylation of the monophenol to odiphenol. This activity distinguishes PPO from other phenol-oxidizing enzymes, such as laccase and peroxidase, and is characterized by the following facts ... 

Laccase contains one type 1 Cu and one type 2 Cu in addition to the type 3 pair. The copper can be reversibly removed from the type 2 site (to give T2D-laccase). Reconstitution may be accomplished by adding CuS04 or Cu1 under anaerobic conditions.958 Loss of type 2 copper has little effect on the redox potentials of the type 1 and type 3 copper, or on the electron-transfer reactivity of the type 1 copper. It appears that type 2 Cu is a substrate-binding site in the reduction pathway for the blue copper.959... 

In the discussion of the biochemistry of copper in Section 62.1.8 it was noted that three types of copper exist in copper enzymes. These are type 1 ( blue copper centres) type 2 ( normal copper centres) and type 3 (which occur as coupled pairs). All three classes are present in the blue copper oxidases laccase, ascorbate oxidase and ceruloplasmin. Laccase contains four copper ions per molecule, and the other two contain eight copper ions per molecule. In all cases oxidation of substrate is linked to the four-electron reduction of dioxygen to water. Unlike cytochrome oxidase, these are water-soluble enzymes, and so are convenient systems for studying the problems of multielectron redox reactions. The type 3 pair of copper centres constitutes the 02-reducing sites in these enzymes, and provides a two-electron pathway to peroxide, bypassing the formation of superoxide. Laccase also contains one type 1 and one type 2 centre. While ascorbate oxidase contains eight copper ions per molecule, so far ESR and analysis data have led to the identification of type 1 (two), type 2 (two) and type 3 (four) copper centres. 

It is possible to prepare R. vernicifera laccase which is reversibly depleted in the type 2 copper. This is a great aid in understanding the role of type 2 copper in the mechanism. Type 2-depleted laccase may exist with the type 3 site in the oxidized or reduced form. The spectral features of the blue type 1 copper change with change in the oxidation state of the type 3 copper. This intersite structural interaction may relate to the electron-transfer pathway between type 1 and type 3 copper.1337 The type 2 site has been implicated in the binding of polyphenolic substrates.959... 

Figure 4.8 Biosynthetic pathway from coniferyl alcohol to (—)-matairesinol. Enzymes and proteins Involved are (1) pinoresinol synthase (laccase + dirigent protein), (2) pinoresinol/lariciresinol synthase, (3) secoisolariciresinol dehydrogenase.

The high-affinity pathway involves oxidation of Fe to Fe by the ferroxidase FET3 and subsequent transport of Fe " " across the plasma membrane by the permease FTRl. FET3p is a member of the family of multicopper oxidases, which include ascorbate oxidase, laccase, and ceruloplasmin (see Chapter 14), and does not become functional until it is loaded with copper intracellularly through the activities of the copper chaperone ATX Ip and the copper transporter CCC2p. It appears that Fe " " produced by FET3 is transferred directly to FTRl, and does not equilibrate with the bulk phase, as is illustrated in Fig. 7.13. This is almost certainly achieved by the classic metabolite-channeling mechanism, a common feature of multifunctional enzymes. 

Figure 8.9 Oxidation mechanism of pyrrole by using the 02/laccase/ABTS biocatalytic pathway (Reproduced with permission from[79]. Copyright (2005) American Chemical Society).

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