# Towards the engineering of fungal laccases for an environmental application. 1st European congress on Engineering Enzymes for biocatalysis by using directed evolution

Claude Jolivalt, Thomas Bertrand, Pierre Briozzo, Catherine Madzak, Nathalie Joly, Eliane Caminade, Christian Mougin

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Claude Jolivalt, Thomas Bertrand, Pierre Briozzo, Catherine Madzak, Nathalie Joly, et al. Towards the engineering of fungal laccases for an environmental application. 1st European congress on Engineering Enzymes for biocatalysis by using directed evolution. 1st European congress on Engineering Enzymes for biocatalysis by using directed evolution, Sep 2002, Paris, France. 2002. [hal-01600362](https://hal.science/hal-01600362)

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<figure><img src="image_1.png" /></figure>
# Towards the engineering of fungal laccases for an environmental application

CLAUDE JOLIVALT<fnref n="1" />, THOMAS BERTRAND<fnref n="2" />, PIERRE BRIOZZO<fnref n="3" />, CATHERINE MADZAK<fnref n="4" />, NATHALIE JOLY<fnref n="3" />, ELIANE CAMINADE<fnref n="5" />, CHRISTIAN MOugin<fnref n="5" />

## INTRODUCTION

- Fungal laccases (EC 1.10.3.2) are a group of multicopper oxidoreductive enzymes involved in lignin transformation, morphogenesis, pathogenesis and fungal virulence, as well as in the oxidation of numerous xenobiotics

- Laccases are widely used in several processes of the dye, paper (pulp bleaching) and textile industries

- A wide range of their substrates are of environmental interest, and are recognized pollutants of waters and soils

- Laccases are potential useful tools to improve the chemical quality of polluted media through bioremediation[¹]

Our objective is the design of laccases with:

- a high efficiency in catalysis (high redox potential)

- a wider variety of substrates

- a pH of activity optimized for natural media

<figure><img src="image_2.png" /></figure>

The present poster reports the preliminary studies intended to allow a better knowledge of the laccase IIb (LacIIb) from the white-rot basidiomycete *Trametes versicolor*, as the first step of its engineering

Our approach is based on the knowledge of the cavity enclosing a weak reducing substrate of the enzyme, the arylamine 2,5-xylidine, and on the replacement of neighboring interacting residues

## RESULTS

### 1 - Expression of LacIIb by *Yarrowia lipolytica* [4]

- The gene has been cloned in *Escherichia coli* using the pGEM®-T plasmid

- It has been sequenced and comprises 1563 bases (sequence accession number AF414109), including a peptide signal

- It was then expressed in the yeast *Yarrowia lipolytica* using the pINA1269 vector (Patent INRA/INA-PG), thus allowing the production of an active form of the recombinant enzyme (transformation of the substrate ABTS to a green oxidized form on solid culture media)

### 2 - Structural data of the wild protein [5]

#### Global fold of LacIIb

(atomic coordinates: pdb accession number 1KYA)

●, copper atoms; ● and O, glycosylation sites

<figure><img src="image_3.png" /></figure>

The crystal structure of the protein has been obtained with 2,5-xylidine bound in its active site

### 3 - T1 site and neighboring 2,5-xylidine ligand

<figure><img src="image_4.png" /></figure>

Two charged or polar residue interact with the amino group of the xylidine:

- His (H458) coordinates the copper that functions as the primary electron acceptor

- Asp (D206) possesses acido-basic properties that could be responsible for the pH-dependent activity towards substrates

untransformed
*Yarrowia*

wild strain of
*Trametes*

*Yarrowia* transformants
expressing laccase

### 5 - Strategy of directed mutagenesis

To a residue with another negative chain:
D206E

To a residue with a polar uncharged chain:
D206N

To a residue with an uncharged chain:
D206A

### 4 - Sequence alignment of laccases from fungi and higher plants

Tversicolor
Ccinereus
Pcinnabarinus
Malbomyces
Athaliana
Rvernicifera
Apseudoplatanus

INGK.GRSPSTTTADLSVISTPFGKRYRFRLOVSLSCDPNYTFSDIDGHNMTIITEDSINTAPLVDSIQTF
INGK.GRYGGPAAELSIVNVEQGKKYRMRLISLSCDPNWQFSIDGHELTIEVDEGELTEPHTVDRLOQIF
INGL.GRTTGAPSDLAVIKVTOGKRYRFRLVSLSCDPNHTFSIDNHTMTIITEAADSINTQPLEVDSIQIF
INGT.AVNPNTGEGQYANVTLTPGKRHRLRILNTSTENHFQVSLVNEHTMTVIAADMVPVNAMTVDSLFLA
INGQ.GDLYNCSTKETVVPIINSGETSLLRVINAAALNQPLFFTVANBKLTVVGADASYLKPFITTKVLMLG
INGELGDLYNCSKETTYRLSVQPNKTYLLRIVNAVLINEEKFFGIAKHTLTVVAQDASYIKPINTSYIMIT
INGQP.GALFDCSTGTTFRLPVKKGETYLLRINAIINLEEMFFGIAQENLRVVGTDFGYTKPINTEYIMIT

- Biological roles of laccases from fungi and higher plants appear to be opposite: fungal laccases are involved in ligninolysis whereas enzymes from plants catalyze the initial polymerization of monolignols during lignification

- Fungal laccases have a negatively charged residue at the 206 position (Asp, D or Glu, E), whilst those of higher plants have an asparagine (Asn, N)

Fungi: Tversicolor = *Trametes versicolor*; Ccinereus = *Coprinus cinereus*; Pcinnabarinus = *Pycnoporus cinnabarinus*; Malbomyces = *Melanocarpus albomyces*

Higher plants: Athaliana = *Arabidopsis thaliana*; Rvernicifera = *Rhus vernicifera*; Apeudoplatanus = *Acer pseudoplatanus*

## CONCLUSIONS

- We describe here the first crystal structure of a laccase complexed with a reducing susbstrate. Relevant residues of the reducing center can thus be localized

- Previously based only on sequence alignment analysis, the directed mutagenesis of laccases has shown only very slight results in improving enzyme efficiency [6]. The present knowledge of structural data will supply new and promising leads

- However, the high redox potential of some fungal laccases remains unexplained

## References

[1] C. Jolivalt, S. Brenon, E. Caminade, C. Mougin, M. Pontié (2000) Immobilization of laccase from *Trametes versicolor* on a modified PVDF microfiltration membrane: characterization of the grafted support and application in removing a phenylurea pesticide in wastewater, J. Membrane Sci. 180:103-113.

[2] C. Mougin, F.-D. Boyer, E. Caminade and R. Rama (2000). Cleavage of the diketonitrile derivative of the herbicide isoxaflutole by extracellular fungal oxidases, J. Agric. Food Chem. 48:4529-4534.

[3] C. Mougin, C. Jolivalt, M. Malosse, J.-C. Sigoillot J.-C., M. Asther and V. Chaplain (2002). Interference of soil contaminants with laccase activity during the transformation of complex mixtures of polycyclic aromatic hydrocarbons (PAH) in liquid media, Polycycl. Aromat. Comp. (in press).

[4] C. Jolivalt, C. Madzak and C. Mougin (2002) Characterization of the gene encoding a laccase of *Trametes versicolor* and analysis of the recombinant enzyme expressed in *Yarrowia lipolytica* (in preparation).

[5] T. Bertrand, C. Jolivalt, P. Briozzo, E. Caminade, N. Joly, C. Madzak and C. Mougin (2002) Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics, Biochemistry 41:7325-7333

[6] F. Xu, R.M. Berka, J.A. Wahleithner, B.A. Nelson, J.R. Shuster, S.H. Brown, A.E. Palmer and E.I. Solomon (1998) Site-directed mutations in fungal laccase: effect on redox potential, activity and pH profile, Biochem. J. 334:63-70.

<footnote n="1">¹Laboratoire de Synthèse Sélective et Produits Naturels, UMR CNRS 7573, ENSCP, 11 rue P. et M. Curie, 75231 Paris Cedex 05,</footnote>

<footnote n="2">²Laboratoire d'Enzymologie et Biochimie Structurales, UPR CNRS 9063, 91198 Gif-sur-Yvette Cedex,</footnote>

<footnote n="3">³Laboratoire de Chimie Biologique, UMR 206 INRA-INAPG, 78850 Thiverval-Grignon,</footnote>

<footnote n="4">⁴Laboratoire de Génétique Moléculaire et Cellulaire, INRA, 78850 Thiverval-Grignon,</footnote>

<footnote n="5">⁵Unité de Phytopharmacie et Médiateurs Chimiques, route de Saint-Cyr, 78026 Versailles Cedex, France,</footnote>

<footnote>E-mail: mougin@versailles.inra.fr</footnote>