1. What are fungal laccases?

Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) catalyze the oxidation of various aromatic, particularly phenolic substrates (e.g. hydroquinone, guaiacol, 2,6-dimethoxyphenol or phenylene diamine), coupled to the reduction of molecular oxygen to water. Laccases as well as ascorbate oxidases (EC 1.10.3.3) and ceruloplasmins (EC 1.16.3.1) usually contain several copper atoms in the catalytic centre. Therefore, they belong to the enzyme superfamily of multicopper oxidases, which is a widely distributed protein family among pro- and eukaryotes. However, laccases or laccase-like multicopper oxidases (LMCO) were predominantly described in fungi and plants, were they occur as multigene family with sometimes more than 10 different laccase genes. For example, the basidiomycete Coprinopsis cinerea and the plant Arabidopsis thaliana have both 17 different genes.

Due to their large capacities to oxidize polyphenols, fungal laccases are involved in several biochemical processes. In the environment they are involved in the biodegradation of lignin and humic acids and biosynthesis of new humic compounds. Thus laccases of litter and wood degrading fungi strongly contribute to soil organic matter turnover and the global carbon cycle. Fungal laccases also play an important role in physiological processes related to pathogenesis, morphogenesis, i.e. fruitbody development, pigmentation and to cell detoxification. While laccases involved in lignin degradation are extracellular enzymes, the ones responsible for the other processes essentially act intracellularly. Due to the oxidation of a variety of substrates, fungal laccases especially from basidiomycetes receive also attention for use in diverse biotechnological applications, like bioremediation. Laccases are involved in the biotransformation of many persistent environmental pollutants, like azo dyes, polycyclic aromatic hydrocarbons, endocrine disruptors or polycyclic musk fragrances.

The catalytic activity of fungal laccases requires a minimum of four copper atoms per active protein (Fig. 1). Accordingly, laccases consist of four regions, which bind the copper atoms of the active centre. These copper binding regions (cbr) are strongly conserved in all laccases (Fig. 2) and were used for the design of degenerate primers to analyze and identify laccase genes of several fungal species or in environmental samples.

More informations about Laccases are available at:

The BRENDA database [link]
The KEGG database [link]
The FOLy database [link]
The BIORENEW program [link]

During my PhD I worked within the DFG joined research program SPP 1090 "Soils as source and sink for CO₂" on the topic “Diversity and expression of fungal and bacterial laccase genes in soils and cultures".

Fig. 2. Amino acid alignment including the four well conserved copper binding regions (cbr I - cbr IV) of laccases used  for the design of degenerate primers and the polyclonal antibody (based on 4 peptides). B - basidiomycetes, A - ascomycetes,
Z - zygomycetes, P - plants.

2. Research questions

Our main questions are of ecological origin. We wanted a molecular tool to identify fungi and assess their functional role in soils. Thus, laccase genes and their involvement in certain degradation processes were a suitable target for this kind of studies. One main aim was to characterize the genetic and functional (“expression”) diversity of fungal laccase genes in a brown forest soil in space and time. Moreover it was interesting to analyze the potential role of bacterial laccase genes. Consequently in future attempts it is necessary to develop proteomic approaches to investigate the role of laccases in soils.
Beside analyzing the role of laccases in the environment, I’m also interested in the genetic diversity, their expression and function in single fungal groups or taxa. Especially ascomycetes are very understudied, and need a focus in future research.

3. Methods of investigation

In order to investigate fungal laccases in pure cultures or environmental samples, standard molecular methods were used. In particular, DNA or RNA (later transcribed in cDNA) was extracted from samples and thereafter used in PCR for amplification of fungal laccase gene fragments. The amplified gene fragments, situated between the conserved copper binding regions (Fig. 3), were cloned and subsequently sequenced. Afterwards the sequences were analyzed for possible introns and aligned together. Then, phylogenetic programs were used to analyze their relationship to fungal reference species or to display the different laccase gene families. Furthermore the expression of laccases in cultures was analyzed, e.g. after induction with naturally occuring phenolic substrates. In recent attempts we try to relate the obtained genetic informations to the appearance of extracellular laccase proteins using proteomic tools like antibody incubation and mass spectrometry of tryptic fragments. 

Fig. 3. Schematic representation of a laccase gene including the conserved copperbinding regions I-IV and the appropriate primer sites for our primers.

Primer name Primer sequence 5’-3’ Primer specificity References Cu1F, Primer I CAY TGG CAY GGN TTY TTY CA basidiomycetes, cultures & soil samples D´Souza 1996; Luis 2004, 2005 Cu1AF ACM WCB GTY CAY TGG CAY GG ascomycetes: cultures & soil RNA (cDNA) bacteria: cultures & soil DNA Kellner 2007, 2008, 2009 Cu2R G RCT GTG GTA CCA GAA NGT NCC fungi and bacteria, cultures & soil samples Luis 2004, 2005; Kellner 2007, 2008, 2009 Cu3R TG ICC RTG IAR RTG IAN IGG RTG fungi, cultures Kellner 2007 Primer II RTG RCT RTG RTA CCA RAA NGT basidiomycetes: cultures D´Souza 1996 LAC2FOR GGI ACI WII TGG TAY CAY WSI CA ascomycetes: cultures & soil DNA Lyons 2003 LAC3REV CC RTG IWK RTG IAW IGG RTG IGG ascomycetes: cultures & soil DNA Lyons 2003 mod1       mod1

4. Results

Within 6 years of study (Patricia Luis & me), it was possible to analyze the spatial and temporal distribution of saprotrophic and mycorrhizal basidiomycete laccase genes in a brown forest soil. Furthermore, a method to investigate their accordingly expression in soil was developed by Patricia Luis and used for the study of an annual cycle. Recent attempts were also successful to analyze the diversity of bacterial laccase genes in soil. In an cooperative attempt we successfully designed and evaluated an antibody against fungal laccases.

see detailed results of our investigations here:

Section I: Spatial distribution and expression of fungal laccase genes in a forest soil [link]
                       work of Patricia Luis, first phase of the SPP1090 project
Section II: Temporal laccase gene diversity and expression in a forest Cambisol [link]
                       work of Harald Kellner, second phase of the SPP1090 project
Section III: Proteomic detection of fungal extracellular laccases [link]
                       work of Harald Kellner & Nico Jehmlich, second phase of the SPP1090 project
Section IV: Spatial distribution of bacterial laccase-like MCOs [link]
                       work of Harald Kellner, second phase of the SPP1090 project

Further: The laccase genes diversity and expression in the Morchellaceae [link]
 

5. Laccase gene detection & analysis in ecosystem research

Within the last years several research projects were done by research groups worldwide using fungal laccase genes as a tool to answer ecological questions in the field. A large focus was to answer the question how increased atmosperic N deposition alters the fungal community (here mainly basidiomycetes), i.e. laccase gene presence, abundance and composition in forest ecosystems. The group around Prof. Zak, University of Michigan used our developed primer pair (Luis et al. 2004) to analyse the abundance of laccase genes using quantitative PCR or length heterogeneity to analyse changes in different forest soils and litter after applications of different levels of NO₃^- (Blackwood et al., 2007; Hofmockel et al., 2007; Hassett et al., 2009; Lauber et al., 2009). The studies were performed in different sites across Michigan with different durations, one short-term study (~6 years of N application; Manistee study) and a long-term study of more than 15 years of N treatment across a wide range of sampling sites (The Michigan Gradient [link]).
A recent study uses laccase genes to investigate the ecosystem response (i.e. fungal response) after repeated fires, which releases multiple nutrients into the soil (Artz et al. 2009; using the primers of D’Souza et al. 1996).

Artz, R.R.E., Reid, E., Anderson, I.C., Campbell, C.D., Cairney, J.W.G., 2009. Long term repeated prescribed burning increases eveness in the basidiomycete laccase gene pool in forest soils. FEMS Microbiology and Ecology 67: 397-410.

Blackwood, C.B., Waldrop, M.P., Zak, D.R., Sinsabaugh, R.L., 2007. Molecular analysis of fungal communities and laccase genes in decomposing litter reveals differences among forest types but no impact of nitrogen deposition. Environmental Microbiology 9: 1306-1316.

Hassett, J.E., Zak, D.R., Blackwood, C.S., Pregitzer, K.S., 2009. Are basidiomycete laccase gene abundance and composition related to reduced lignolytic activity under elevated atmospheric NO₃^- deposition in a Northern Hardwood Forest? Microbial Ecology 57: 728-739.

Hofmockel, K.S., Zak, D.R., Blackwood, C.B., 2007. Does Atmospheric NO₃^- Deposition Alter the Abundance and Activity of Ligninolytic Fungi in Forest Soils? Ecosystems 10: 1278–1286.

Lauber, C.L., Sinsabaugh, R.L., Zak, D.R., 2009. Laccase gene composition and relative abundance in Oak forest soil is not affected by short-term nitrogen fertilization. Microbial Ecology 57: 50-57.

6. Suggested readings

Baldrian P (2006) Fungal laccases: occurrence and properties. FEMS Microbiol Rev 30: 215-242.

D’Souza TM, Boominathan K & Reddy CA (1996) Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR. Appl Environ Microbiol 62: 3739-3744.

Hoegger PJ, Kilaru S, James TY, Thacker JR & Kües U (2006) Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences. FEBS J 273: 2308-2326.

Kellner H., Luis P., Zimdars B., Kiesel B., Buscot F. : Diversity of bacterial laccase-like multicopper oxidase genes in forest and grassland Cambisol soil samples. Soil Biol Biochem 40: 638-648.

Kellner H., Jehmlich N., Benndorf D., Hoffmann R., Rühl M., Hoegger P.J., Majcherczyk A., Kües U., von Bergen M., Buscot F. (2007): Detection, quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry. Enzymes and Microbial Technology 41: 694-701.

Kellner H., Luis P., & Buscot F. (2007) Diversity of laccase-like multicopper oxidase (LMCO) genes in Morchellaceae: identification of genes potentially involved in extracellular activities related to plant litter decay. FEMS Microbiol Ecol 61: 153-163.

Kellner H., Luis P., Schlitt B., Buscot F. (2009): Temporal changes in diversity and expression patterns of fungal laccase genes within the organic horizon of a brown forest soil. Soil Biol Biochem 41: 1380-1389.

Luis P, Walther G, Kellner H, Martin F & Buscot F (2004) Diversity of laccase genes from basidiomycetes in a forest soil. Soil Biol Biochem 36: 1025-1036.

Luis P, Kellner H, Martin F & Buscot F (2005a) A molecular method to evaluate basidiomycete laccase gene expression in forest soils. Geoderma 128: 18-27.

Luis P, Kellner H, Zimdars B, Langer U, Martin F & Buscot F (2005b) Patchiness and spatial distribution of laccase genes of ectomycorrhizal, saprotrophic and unknown basidiomycetes in the upper horizons of a mixed forest Cambisol. Microb Ecol 50: 570-579.

Lyons JI, Newell SY, Buchan A & Moran MA (2003) Diversity of ascomycete laccase gene sequences in a Southeastern US salt marsh. Microb Ecol 45: 270-281.

Piontek K, Antorini M, Choinowski T (2002). Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-A resolution containing a full complement of coppers. J Biol Chem 277: 37663-37669.

Acknowledgement

I have to thank F. Buscot, P. Luis, B. Zimdars, N. Jehmlich, M. Pecyna for their help as well as many more people.

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