Laccases from tree

Laccase was first isolated by Yoshida in 1883 [43] from tree lacquer of Rhus ver-nicifera. Laccases can thus be classified according to their source plant, fungal or, more recently, bacterial or insect [44], The laccase enzyme active site contains four copper ions classified into three types based upon their geometry and coordinating ligands, denoted... 

Eckenrode et al. demonstrated that copper oxidases could oxidize vindoline through the same sequence of intermediates found in the metabolism of the alkaloid by Streptomyces griseus. Laccase from Polyporus anceps, laccase from the lacquer tree, and the mammalian (human serum) equivalent copper oxidase... 

The mechanism of dioxygen reduction at the trinuclear cluster in MCO catalysis has been a strong focus for research on this class of copper oxidases. Dioxygen reduction has been most thoroughly investigated in Rhus laccase (a plant laccase, from the Japanese lacquer tree). The primary reason for using Rhus Lac is the availability of a metal-substituted form the enzyme, a TlHg form, in which the... 

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. 

Redox potentials for the different copper centers in the blue oxidases have been determined for all members of the group but in each case only for a limited number of species. The available data are summarized in Table VI 120, 121). The redox potentials for the type-1 copper of tree laccase and ascorbate oxidase are in the range of 330-400 mV and comparable to the values determined for the small blue copper proteins plastocyanin, azurin, and cucumber basic protein (for redox potentials of small blue copper proteins, see the review of Sykes 122)). The high potential for the fungal Polyporus laccase is probably due to a leucine or phenylalanine residue at the fourth coordination position, which has been observed in the amino-acid sequences of fungal laccases from other species (see Table IV and Section V.B). Two different redox potentials for the type-1 copper were observed for human ceruloplasmin 105). The 490-mV potential can be assigned to the two type-1 copper sites with methionine ligand and the 580-mV potential to the type-1 center with the isosteric leucine at this position (see Section V.B). The... 

A ping-pong di Theorell-Chance mechanism has been deduced for tree laccase from steady-state kinetics (123). This mechanism is characterized by the sequential entry of the two substrates and the immediate... 

Faced with the problem of elucidating the individual roles of the diflFerent copper centers in the blue oxidases, the researcher has naturally focused in recent years on the laccases (9). Being easier to isolate, better characterized, and containing fewer copper atoms than cemloplasmin or ascorbate oxidase, the laccases from the Japanese lacquer tree Rhus vernicifera and the fungus Polyporus versicolor have been the subject of several transient kinetic studies in the millisecond range, that is, studies using stopped-flow spectrophotometry and rapid-freeze EPR spectroscopy (9,49,50). 

A two-site ping pong bi bi mechanism has been deduced for tree laccase from steady-state kinetics. This will be valid for ascorbate oxidase as well because both enzymes are structurally and mechanistically closely related. A reaction scheme for ascorbate oxidase has been proposed based on the available spectroscopic, kinetic, and structural information (Figure 35) (Messerschmidt ) that should also be valid for laccase or... 

Table 5.2 contains data about selected copper enzymes from the references noted. It should be understood that enzymes from different sources—that is, azurin from Alcaligenes denitrificans versus Pseudomonas aeruginosa, fungal versus tree laccase, or arthropodan versus molluscan hemocyanin—will differ from each other to various degrees. Azurins have similar tertiary structures—in contrast to arthropodan and molluscan hemocyanins, whose tertiary and quaternary structures show large deviations. Most copper enzymes contain one type of copper center, but laccase, ascorbate oxidase, and ceruloplasmin contain Type I, Type II, and Type III centers. For a more complete and specific listing of copper enzyme properties, see, for instance, the review article by Solomon et al.4... 

The elaborate mechanism by which blue oxidases react with dioxygen to produce water was tackled by studying the possible role of H202. We have observed the formation of a stable and high affinity complex between tree laccase and H202. Moreover, the finding that the oxidation of the reduced enzyme with H202 follows a pattern which is different from that operative in the reduction of the oxidized enzyme may have important implications for the mechanism of action of laccase. 

Rusticyanin has a high reduction potential (680 mV), which is similar to that for the Type 1 Cu center in fungal as opposed to tree laccase (785 mV) (73). This trend is so far unexplained. From the sequence and EXAFS studies, His-Cys-His-Met coordination is a reasonable possibility for rusticyanin (55). It may well be that the reduction potential is determined by effects of a polypeptide backbone on Cu—S(Cys) and Cu—S(Met) bond distances and the Cu ligand field (74). If this is the case, however, rusticyanin would be expected to have one or both Cu—S distances shorter than in other blue copper proteins, which is not borne out by information from EXAFS (Table IV). A further possibility that the Cu(I) form is three-coordinate, as in the case of plasto-cyanin at low pH (Fig. 2), has no strong support at present (75). 

In contrast to tree and fungal laccase, whose molecular parameters and mechanisms of action have been thoroughly investigated (8), few such studies have been reported for ascorbate oxidase. This is mainly because of the relatively diflScult isolation and purification procedure of ascorbate oxidase in comparison with laccase. Furthermore, this enzyme appears to be more sensitive to environmental factors such as ionic strength of the buffer medium, its pH, or the presence of extraneous metal ions. Consequently, many samples isolated over a long period were found to be homogeneous from the standpoint of the protein biochemist but appeared inhomogeneous with respect to the catalytically active copper sites (9). 

The finding from rapid-freeze-quench EPR experiments, that the reduction of the type 2 copper is slow compared with that of the type 1 copper, is analogous to the behavior noted for tree laccase at higher pH values (50). In this enzyme the slow reduction of the type 2 center is linked to the inhibition of the type 3 reduction. In ascorbate oxidase, however, reduction of the type 3 copper pairs proceeds despite the slow reduction of the type 2 copper, suggesting that the two electrons necessary for the proposed intramolecular reduction of the two type 3 copper pairs can be transferred from two of the three type 1 copper centers, without involving the type 2 center in any redox process. 

Type 2 copper directly or indirectly via the Type 1 copper in a fast intramolecular process. The second phase of the reaction involves simultaneous transfer of two electrons from the reduced Types 1 and 2 to the oxidized Type 3 binuclear copper center. The final phase involves rereduction of the Types 1 and 2 to the oxidized Type 3 binuclear copper center. The final phase involves rereduction of the Types 1 and 2 copper. Thus electrons appear to be transferred through the enzyme in pairs which accounts for the lack of EPR detectable species and n=2 Nemst behavior of the Type 3 center under most experimental conditions. A detailed study of the interaction of NO with both fungal and tree laccase has recently appeared and is generally consistent with the above scheme . 

Laccase has been isolated from lacquer trees (e.g. Rhus vernifera) and from various fungi. The crystal structure of... 

The name laccase was first given to the enzyme by G. Bertrand (46) who studied its activity in crude form from the latex of the lac tree Rhus succedanea, and similar enzymes have been found in the latex of various other Asian lac trees... 

Laccases do not possess hydroxylating properties. They oxidize o-diphenols and p-diphenols by a radicalic mechanism. Enzymes of this type were first obtained from the Japanese lack tree Rhus vernicifera. Laccases contain 4 atoms of copper 2 Cu+ ions and 2 Cu + ions. One of the latter is responsible for the blue color of the enzymes. This Cu + ion is reduced by the substrate to Cu+, i.e., it has the properties of an electron carrier (as is the case with iron in dioxygenases and some monooxygenases, C 2.5 and C 2.6). Hence laccases are capable of producing radicals from phenols according to the following equation ... 

Laccase. A polyphenol oxidase has been purified from the sap of the lac tree by Keilin and Mann. Laccase differs from the potato and mushroom enzyme in several respects. With regard to substrate specificity, it oxidizes p-phenylenediamine more rapidly than catechol. p-Phenylene-diamine is not a substrate for the other polyphenol oxidases described. Laccase apparently is inert with p-cresol. It is not inhibited by carbon monoxide. Unlike the other phenol oxidases, this enzyme is not a pale yellow, but is blue, as is ascorbic acid oxidase (see below). This enzyme, however, is not an ascorbic acid oxidase. 

The first paper mill was buUt by Eiichi Shibusawa in Tokyo in 1872. Some tons of natural rubber were imported from India and the USA in 1880-1890 and, concurrently, a rubber manufacturing company was buUt (1886). Celluloid products were first imported from Germany in 1877, and their domestic production started around 1890. The most interesting example is the Japanese urushi lacquer made from the poison oak tree. Because of its bright and clear color, the lacquer has been widely used from commodity to art works in Japan. In 1883, Hrkorokuro Yoshida published a research article on the urushi lacquer, describing an enzyme (later named laccase) that initiated the polycondensation of urushiol [5]. After 1903, scientific study on the urushi lacquer was continued by RUcou Majima [6]. 

Laccase has been isolated from lacquer trees (e.g. Rhus vernifera) and from various fungi. The crystal structure of laccase obtained from the fungus Trametes versicolor was reported in 2002 and confirms the presence of a trinuclear copper site containing Type 2 and Type 3 copper atoms, and a monocopper (Type 1) site. The structure of the trinuclear copper site is similar to that in ascorbate oxidase (Fig. 29.14). However, the Type 1 copper atom in laccase is 3-coordinate (trigonal planar and bound by one Cys and two His residues) and lacks the axial ligand... 

The sap of the lacquer trees is the material for Japanese lacquer and is collected from the lacquer trees, Rhus vernicifera, as a latex as a water in oil (urushiol) type emulsion. This sap consists of urushiol (65-70%)(see Fig. 1), plant gum (5-7%), laccase (less than 1%) and water (20-25%). 

---