«by CHIEH-TING WANG (Under the Direction of Jeffrey F. D. Dean) ABSTRACT Laccase and related laccase-like multicopper oxidases (LMCOs) have been ...»
1994). The purified phenoloxidase was later confirmed as a LMCO (Alexandre and Bally 1999, Diamantidis et al. 2000).
Melanization related to a bacterial LMCO has also been observed in the marine bacterium, Marinomonas mediterranea (Solano et al., 2000), but no functional role has yet been assigned to this enzyme.
A laccase-like enzyme activity was found in spores of a Bacillus sphaericus strain (Claus and Filip 1997). The spore protein, CotA, of Bacillus subtilis has also been recognized to be a LMCO (Hullo et al. 2001). Mutants in the gene encoding CotA lost the ability to produce a brownish spore pigment. The oxidation of Mn2+ by laccase-like spore proteins of a marine Bacillus strain has been shown from mutants in genes coding for multi-copper oxidases that had lost their metal-oxidizing activities (van Waasbergen et al. 1996). The involvement of similar enzymes in manganese oxidation by various Pseudomonas species has also been suggested (Brouwers et al. 1999, Francis and Tebo 2001). The yacK gene product of E. coli has features typical of a MCO and has ferroxidase activity (Kim et al. 2001).
pseudoazurin domains in conjunction with a Type-1 Cu2+ in domain 3 and a trinuclear Cu2+ cluster between domains 1 and 3 (Roberts et al. 2002). The authors suggested a role for the enzyme in the uptake of and tolerance to copper and iron cations.
Fungal LMCOs LMOCs from fungi are the most diverse and extensively studied members of this class of enzymes. The large number of fungi producing LMCOs and the diversity of LMCO isoforms they produce suggest that these enzymes play a host of important roles in fungal biology (Thurston 1994, Mayer and Staples 2002). The involvement of LMCOs in fungal morphogenesis is probably the best established of the numerous putative functions given for these enzymes. Biochemical and genetic evidence that a LMCO gene product in Aspergillus nidulans catalyzes the final step in spore pigment formation, the conversion of a yellow polyketide precursor into a dark green, polymeric product, suggests that the LMCO is important for structural development of fungal spores (Law and Timberlake 1980, Aramayo and Timberlake 1990).
LMCOs have also been proposed to participate in fungal morphogenesis in Armillaria spp. (Worral et al. 1986), Lentinus edodes (Leatham and Stahmann 1981) and Volvacea volvacea (Chen et al. 2004).
by certain white-rot fungi, although the presence of both peroxidases and LMCOs has been an obstacle in elucidating their precise roles in lignin biodegradation (Ranocha et al. 2000).
Many ligninolytic fungi, such as Phlebia radiata, Phanerochaete chrysosporium, and Rigidiporus lignosus secrete both classes of enzyme. Pycnoporus cinnabarinus, however, has become a model species to demonstrate the role of LMCOs in ligninolysis since it produces LMCOs only, and not any of the peroxidases (lignin peroxidase and manganese peroxidase), commonly associated with lignin degradation (Eggert et al. 1996). This fungus is perfectly capable of hydrolyzing lignin polymers under normal conditions, whereas a LMCO-deficient P. cinnabarinus mutant was unable to hydrolyze synthetic lignin (DHP) polymers (Eggert et al. 1997).
The reaction mechanism by which LMCOs catalyze lignin degradation is not completely understood. Studies indicate that LMCOs may oxidize the terminal phenolic units in lignin, but they are unable to directly oxidize non-phenolic lignin substructures due to their low redox potentials (Eriksson et al.
1990). Thus, small organic compounds or metals that can be oxidized and activated by fungal LMCOs, e.g., veratryl alcohol (Nishizawa et al. 1995), 3-hydroxy-anthranilic acid (Eggert et al. 1996), or Mn2+ (Schlosser and Hofer 2002), have been proposed
lignin structures. Non-physiological redox-mediators have also been used in biotechnological processes to increase the oxidation potential of LMCOs for similar purposes (Li et al.
LMCOs of various fungi may also be involved in virulence mechanisms (Thurston 1994, Mayer and Staples 2002). For example, the grapevine grey mould, Botrytis cinerea, produces a LMCO that is necessary for pathogenesis, and the role of this LMCO is suggested to be in detoxifying antifungal metabolites produced by the host (Bar-Nun et al. 1988). LMCOs are also important for pathogenesis in the chestnut blight fungus, Cryphonectria parasitica, and the human pathogen, Crytococcus neoformans (Mayer and Staples 2002). In C. neoformans, synthesis of melanin, an important virulence factor for this fungus, was shown to be dependent on a single, copper-dependent LMCO (CNLAC1), a role confirmed using knock-out strains of the fungus (Salas et al. 1996, Williamson et al. 1998). In mice, however, Liu et al. (1999) suggested that the ferroxidase activity of this LMCO may protect C. neoformans from macrophages by oxidizing phagosomal iron to Fe3+ with a resultant decrease in antifungal hydroxyl radical formation.
The occurrence of LMCOs in plants has for a long time appeared more limited than in fungi. The first laccase was identified in sap from Rhus vernicifera, the Japanese lacquer tree, by Yoshida (1883). Since then, LMCOs have been described in various Rhus species (Keilin and Mann 1939, Nakamura 1958) and a few other plant species (Mayer and Staples 2002, McCaig et al. 2005). Cell suspension cultures of sycamore maple (Acer pseudoplatanus) were shown to produce and secrete a LMCO (Bligny and Douce 1983, Driouich et al. 1992, Sterjiades et al. 1992), and loblolly pine (Pinus taeda) was shown to produce at least eight LMCOs, all expressed predominantly in differentiating xylem (Sato et al. 2001). Five distinct LMCOs were shown to be expressed in the xylem tissues of poplar (Populus trichocarpa) (Ranocha et al. 1999), and four closely related LMCOs were identified in xylem tissues of yellow-poplar (Liriodendron tulipifera) (LaFayette et al. 1999). LMCOs have also been reported in other species, including Zinnia elegans (Liu et al.
1994), tobacco (Nicotiana tabacum)(Kiefer-Meyer et al. 1996), peach (Prunus persica)(Mayer and Staples 2002), and Sitka spruce (Picea sitchensis) (McDougall 2000). Recently, McCaig et al.
(2005) used LMCO sequences culled from GenBank and the Arabidopsis thaliana genome to construct a gene phylogeny that clearly divided plant LMCOs into at least six distinct classes
widespread amongst angiosperms, and quite probably in gymnosperms as well.
Despite the fact that LMCOs were first discovered in plants over 100 years ago, relatively little is known about the role these enzymes play in vivo. Many plant LMCOs appear to be cell wall-associated enzymes that are relatively difficult to purify.
Consequently, solid biochemical and molecular data are difficult to come by (Ranocha et al. 1999). Wound healing through oxidative polymerization of alkylcatechols in lacquer tree sap (true laccase) had long been the only strongly supported physiological function advanced for plant LMCOs (Mayer and staples 2002; McCaig et al. 2005). The sap from Rhus is used for a wide variety of purposes in Asia, including the manufacture of ornamental lacquerware in Japan, and consequently its production is of considerable economic importance (Dean and Eriksson, 1994).
Plant LMCOs have also been proposed to play a part in lignin biosynthesis (Dean and Eriksson 1994; Dean et al. 1998; Mayer and Staples 2002). Freudenberg and co-workers were the first to suggest a role for laccase in lignification based on studies using a fungal LMCO (Freudenberg, 1950; Freudenberg and Dietrich, 1953; Freudenberg, 1959). Later, however, Nakamura (1967) claimed that such enzymes were likely not responsible for
lacquer tree sap could not polymerize the lignin precursor, coniferyl alcohol. Moreover, a histochemical study using syringaldazine demonstrated peroxidase, but not LMCO, activity in lignifying xylem from a variety of tree species (Harkin and Obst 1973). On the basis of these two negative results, LMCOs were dropped from consideration as oxidative catalysts for lignification, and peroxidases were proposed as the sole enzyme responsible for lignin polymerization (O’Malley et al. 1993, Olson and Varner 1993).
In the early 1990’s, characterization of the LMCO purified from cell-suspension cultures of sycamore maple prompted a reevaluation of the role this enzyme might have in lignification (Bligny and Douce 1983, Sterjiades et al. 1992, Driouich et al.
1992). Sterjiades et al. (1993) showed that the LMCO purified from sycamore maple cells was capable of oxidizing the lignin precursors, or monolignols (sinapyl alcohol, coniferyl alcohol, or p-coumaryl alcohol), to form water-insoluble dehydrogenation polymers (DHPs). In addition, a LMCO isolated from loblolly pine xylem was shown to produce DHPs from coniferyl alcohol, and the activity was localized to lignifying xylem near the cambium, whereas peroxidase activity was quite evident throughout pine stems and was not specifically localized to tissues undergoing active lignification (Bao et al. 1993). Similarly, a tight
cell walls in developing primary xylem was also observed using histochemical staining (Liu et al. 1994). These studies clearly showed that LMCO activity is spatially and temporally associated with lignification in plants. It is possible that, as suggested by Sterjiades et al. (1993), LMCOs and peroxidases are separated temporally in their roles as catalysts for lignin deposition.
However, transgenic plants with down-regulated LMCOs showed no detectable alteration in lignin content or composition of xylem tissues (Dean et al. 1998, Ranocha et al. 2002). Thus, whether plant LMCOs are direct catalysts for lignin biosynthesis remains unclear.
On the basis of specific enzyme activities associated with plant LMCOs, some other putative functions have been discussed in different reports. Recent demonstration of ferroxidase activity in a LMCO from yellow-poplar (Hoopes and Dean 2004) was taken to suggest that some plant LMCOs might play a role in iron metabolism, similar to the function provided by the Fet3 LMCO in the high-affinity iron uptake system described previously. The plausibility of this argument is enhanced by widespread ferroxidase activity in LMCOs from fungi, such as Cryptococcus neoformans (Liu et al. 1999) and Phanerochaete chrysosporium (Larrondo et al. 2003), and bacteria, E. coli (yacK/CueO) (Kim et al. 2001, Singh et al. 2004), as well as the green alga,
al. 2002). Based on widespread distribution of MCOs involved in iron metabolism in all kingdoms, it is reasonable to think that some LMCOs in plants may take part in iron uptake systems similar to the yeast Fet3 system. LMCOs are also one of several oxidative enzymes with demonstrated capacity to degrade chemical compounds in their immediate vicinity (Boyajian and Carreira 1997). That transgenic Arabidopsis over-expressing a secretory LMCO from cotton (Gossypium arboretum) exhibited enhanced resistance to several phenolic allelochemicals and 2,4,6trichlorophenol suggests that plant LMCOs could also play a role in detoxification of organic compounds (Wang et al. 2004). Most of the LMCOs studied in plants can oxidize phenolic compounds and their derivatives, which are related to compounds involved in developmental processes, defense, and signaling. Thus, LMCOs have the potential to play a host of roles in whole plants.
However, in most cases, definitive demonstration of physiological functions for plant LMCOs has been difficult because the enzymes are typically able to oxidize a wide variety of phenolic and inorganic substrates. In addition, multiple LMCO gene products may be expressed simultaneously in the same tissues. To overcome these difficulties, and to distinguish LMCO from peroxidase function, the use of combined biochemical and molecular approaches must be applied. Ranocha et al. (1999) were
LMCO cDNAs and performing biochemical characterization of one of the corresponding gene products.
Structure and catalytic mechanism of LMCOs Structural studies of LMCOs have so far been limited to fungi, as crystal structures of LMCOs from three fungal species have been determined (Ducros et al., 1998; Piontek et al., 2002;
Bertrand et al., 2002; Hakulinen et al. 2002). The protein fold structure of these LMCOs consists of three cupredoxin-like domains (A, B, and C) (Figure 1.3, Hakulinen et al. 2002), which have also been found in ascorbate oxidase (Messerschmidt et al.
1992) and ceruloplasmin (Murphy et al. 1997). All three domains are important for the catalytic activity of LMCOs. There are significant differences in the loops forming the substratebinding pocket in different LMCOs, suggesting different affinities for different substrates (Hakulinen et al. 2002).
The substrate-binding site is located in a cleft between domains B and C, close to the mononuclear copper center in which the Type-1 copper atom is coordinated to two histidine and one cysteine residues.
Multiple sequence alignments of the fungal LMCOs available in public databases have identified distinctive sequence signatures around the conserved copper-binding domains that distinguish fungal LMCOs from plant LMCOs and other multicopper oxidases. The
segments, L1 to L4, that contain the 12 residues serving as copper ligands (Figure 1.4) (Kumar et al. 2003). These four blocks can be found in all MCO amino acid sequences. Two of these regions are near the C-terminus, separated by 35-75 amino acid residues. The other two are near the N-terminus and separated by 35-60 residues (Brouwers et al. 2000, Kumar et al.