Comprehensive Technology Information

Multi-Copper Oxidase Families

Multicopper oxidases (MCOs) family, a group of polyphenol oxidases, has a three-domain structure and usually contains four copper atoms. MCOs are characterized by the three spectroscopically different copper binding sites. They are T1 with one copper atom, T2 with also one copper atom and T3 with two copper atoms. T2 and T3 organize into a single copper cluster. Two histidines and one cysteine serve as ligands for copper at T1 and eight histidines for copper binding at the T2/T3 cluster. The ten histidines and the one cysteine are obviously important residues within the copper-binding domains. Besides, the three-dimensional organization of three-domain MCOs is highly conservative despite regions of considerable sequence divergence.

Multi-Copper Oxidase Families

MCOs can have a distinct diphenol activity mainly catalyze aromatic compounds, while other MCOs have other activities. Applications of MCOs can range from pulp delignification, textile dye bleaching and water or soil detoxification, to the formation of pigments, the development of clinical tests and applications in the field of biosensors, bioreactors, and biofuel cells.

The MCOs from basidiomycete, ascomycete and agaricomycete have been studied much by phylogenetic analysis. The number of MCO coding genes present in ascomycetous, especially in A. niger, is the highest reported so far, and higher than those reported in basidiomycetes, with the exception of coprinopsis cinerea which belongs to agaricomycetes and contains 17 MCO coding genes. Distinct MCO genes and their coding products have been further characterized in these species. MCOs are clustered into different groups, which reveal a number of interesting issues. The first large cluster of MCOs is Laccase. Other members of these subfamilies are ferroxidases, ferroxidases/laccases, bilirubin oxidases, ascorbate oxidase and fungal pigment MCOs.

Laccases (sensu stricto) subfamiliy

Laccases form the largest subgroup within the MCO family which attracts the most attention in biotechnology studies. Laccases sensu stricto are possibly specific to the Agaricomycetes. Laccases catalyze the reduction of oxygen to water accompanied by the oxidation of a substrate, typically a p-dihydroxy phenol or another phenolic compound. In other words, it promotes the following biochemical reaction under transfer of four electrons from the organic substrate to molecular oxygen: 4 benzenediol + O2= 4 benzosemiquinone + 2 H2O. Natural compounds as potential mediators to function upon laccase activation, include 3-hydroxyanthranilate, 4-hydroxybenzoid acid, 4-hydroxybenzyl alcohol, phenol, aniline, vanillin, acetovanillone, methyl vanillate, syringaldehyde, acetosyringone, and p-coumaric acid.

As extracellular enzymes, purification procedures of laccases are very easy to be carried out. And additionally, laccases generally exhibit a considerable level of stability in the extracellular environment. In terms of enzymes functions, Agaricomycetes laccases sensu strictoin play a role in recalcitrant lignocellulosic substrate degradation.

Fungal ferroxidases (Fet3-type)

Fungal ferroxidases include basidiomycete and ascomycete MCOs. Most basidiomycete species contain genes for canonical Fet3-type ferroxidases. These fet3 candidate genes appear often to cluster with a gene encoding Ftr1-related potential iron permease, which is in accordance with the mechanism found in ascomycetous yeast. That is, Fet3 and the specific iron permease Ftr1 form a protein complex at the plasma membrane, in order to mediate the transport of Fe3+ into the cells. Besides, aspergillus niger MCOs, McoH and McoK, also belong to this subfamily, which are both 49% identical to Fet3 protein, and responsible for the reductive iron assimilation system with high affinity iron transport ability.


The large cluster of ferroxidases/laccases contains one of the ascomycete MCOs, aspergillus niger McoE with strong ferroxidase and weak laccase activities. Another four basidiomycete enzymes are also included. They are Mco1 and Phanerochaete flavido-alba PfaL, Mco6 (CnLac1) and Mco5 (CnLac2), with both ferroxidase and laccase activities, but different in strength. Among this large cluster, Mco1 can efficiently oxidize iron and aromatic amines, but not phenolic compounds. PfaL oxidizes a range of organic substrates including aromatic amines. Specific residues (E185, D283, Y354, D409) at four different protein regions are known to contribute to catalysis of Fe2+, in which the former three residues are decisive for oxidation process as signature motifs of multicopper ferroxidases.

Bilirubin oxidases

Bilirubin oxidases (BOD), with four histidine-rich copper-binding domains characteristic of MCOs, catalyzes the chemical reaction 2 bilirubin + O2 = 2 biliverdin + 2 H2O, utilizing four Cu+/2+ ions. Thus, the two substrates of this enzyme are bilirubin and O2, whereas its two products are biliverdin and H2O.

BODs have attracted a lot of attention for the reduction of O2 under physiological conditions. Unlike laccases, these enzymes display a high activity and stability at neutral pH, a high tolerance towards chloride anions and other chelators, and for some species, a high thermal tolerance. In the medical field, the detection of bilirubin is of particular importance, which makes BODs attractive enzymes in that respect.

Ascorbate oxidases

Ascorbate oxidase, originally named hexoxidase, belongs to the blue oxidases of multicopper enzyme, which catalyzes the four-electron reduction of molecular oxygen to water with concomitant one-electron oxidation of the substrate. There are indications for plant ascorbate oxidases to act in oxygen homeostasis and ROS (reactive oxygen species) balancing, in various stress reactions, in defence, in growth and cell wall formation, and in signaling.

Ascorbate oxidase was not typically present only in plant tissues until thermostable enzyme ASOM was discovered, which is the only one that has been characterized as fungal ascorbate oxidase with no laccase activity.

Fungal pigment MCOs

The last cluster mentioned here is named by an enzyme YA from aspergillus nidulans that converts a yellow precursor into a green pigment which gives the aspergillus nidulans conidia their typical color. The other one in this cluster is Abr2p from aspergillus fumigatus, which is also involved in conidial pigment biosynthesis. Both of them are reported to have laccase activity.

As stated above, we can basically know about the multicopper oxidases family and their main members. There are still much more work to do to unravel various MCOs functions using new modern molecular, biochemical and cytological techniques.

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