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Purification, crystallisation and X-ray diffraction study of fully functional laccases from two ligninolytic fungi
Biochimica et Biophysica Acta 1594 (2002) 109^114 www.bba-direct.com
Puri¢cation, crystallisation and X-ray di¡raction study of fully functional laccases from two ligninolytic fungi Matteo Antorini a , Isabelle Herpoe«l-Gimbert a , Thomas Choinowski a , JeanClaude Sigoillot b , Marcel Asther b , Kaspar Winterhalter a , Klaus Piontek a; * b
a Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Universita«tstrasse 16, CH-8092 Zu«rich, Switzerland Unite¨ INRA de Biotechnologie des Champignons Filamenteux, INRA, IFR 86 BAIM, Universite¨s de Provence et de la Me¨diterrane¨e, Faculte¨ des Sciences de Luminy, ESIL, 163 Avenue de Luminy, Case Postale 925, F-13288 Marseille Cedex, France
Received 18 July 2001; accepted 21 September 2001
Abstract Laccase isozymes from the white-rot basidiomycete fungi Trametes versicolor and Pycnoporus cinnabarinus were purified to apparent iso-electric homogeneity and crystallised. T. versicolor laccase crystallises in two crystal forms, both with the î resolution, respectively. The crystals of P. cinnabarinus orthorhombic space group P21 21 21 , which diffract to 1.9 and 2.95 A î resolution. All the laccase crystals are suitable laccase belong to the monoclinic space group C2 and diffract to at least 2.2 A for X-ray structure determination and contain a full complement of copper ions. ß 2002 Elsevier Science B.V. All rights reserved. Keywords: Laccase; Micro-heterogeneity; Crystal; Structure; Fungus
Laccase (benzenediol oxygen oxidoreductase, EC 1.10.3.2), a polyphenol oxidase belongs to the family of blue multi-copper oxidases. Laccases oxidise a broad range of substrates such as polyphenols, methoxy-substituted phenols, diamines and even some inorganic compounds [1^3]. The one-electron oxidation of these reducing substrates occurs concomi-
Abbreviations: ABTS, 2,2P-azino-bis-[3-ethylthiazoline-6sulphonate] ; Lac I and Lac II, laccase isozymes I and II, respectively, from Pycnoporus cinnabarinus; IEF, iso-electric focusing; LiP, lignin peroxidase; MnP, manganese peroxidase; RT, room temperature; SDS^PAGE, sodium dodecyl sulphate^polyacrylamide gel electrophoresis ; TvL, laccase from Trametes versicolor * Corresponding author. Fax: +41-1-632-1121. E-mail address: [email protected] (K. Piontek).
tantly with a four-electron reduction of molecular oxygen to water (for a review see [4]). The laccase enzyme, which was ¢rst discovered more than one century ago in the Japanese tree Rhus venicifera [5], is widely distributed among plants, where it is implicated in wound-response and the synthesis of lignin, the complex polymer that constitutes a main component of the plant cell wall [6]. In contrast, the laccases produced by some wood-rotting fungi from the genus basidiomycete play an important role in the degradation of lignin. The laccases, and two peroxidases produced by the ligninolytic system of some white-rot fungi, lignin peroxidase (LiP) and manganese peroxidase (MnP) are the extracellular enzymes, which can substantially depolymerise lignin [7]. While LiP cleaves the nonphenolic aromatic bonds of lignin, MnP and the fun-
0167-4838 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 1 ) 0 0 2 8 9 - 8
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gal laccases can oxidise only the phenolic components of lignin. In the presence of synthetically produced mediators such as 2,2P-azino-bis-[3-ethylthiazoline-6-sulphonate] (ABTS), the oxidising capability of the laccases is expanded to include the oxidation of non-phenolic compounds [8], and unlike the ligninolytic enzymes LiP and MnP, which require hydrogen peroxide for activation, the laccases are oxidised by O2 . The possibility to extend the substrate range of laccases in the presence of speci¢c mediators and the ability of some fungal laccases to degrade recalcitrant aromatic compounds with high redox potentials, gives this class of enzymes great potential value to the development of environmentally safe processes in the paper and pulp industries [9] as well as in the bioremediation of soils and water. An obstacle that still has to be overcome is that the mechanism by which the laccases attain their speci¢c redox potential is only poorly understood [1,2]. A three-dimensional structure of this enzyme may help to clarify this issue. Despite the many e¡orts to crystallise laccase and the fact that it has been under investigation for many decades, a crystal structure of laccase was reported only recently [10]. Unfortunately, this Coprinus cinereus laccase is of a Cu-depleted form, in which the putative type-2 Cu is completely absent. Trametes versicolor and Pycnoporus cinnabarinus are typical white-rot fungi that have been extensively investigated due to their ligninolytic capability [11]. The P. cinnabarinus laccase gene has been cloned and expressed in Pichia pastoris [12] and more recently in Aspergillus niger. The active recombinant form of laccase was expressed, opening the way for site-directed mutagenesis. This, in combination with structural studies, will give us more insight into the functionality of this enzyme. We report the ¢rst successful crystallisation of fully active and fully glycosylated laccases containing a full complement of coppers. Precultures of T. versicolor (ATCC 20869) were obtained by inoculating mycelial fragments into a glass Erlenmeyer £ask containing 250 ml of a medium prepared according to Fahreus and Reinhammar [13]. Several glass marbles were added to disrupt the mycelium. The culture medium was incubated at room temperature (RT) on a rotary shaker for 0.5 h
and subsequently allowed to grow for 24 h without shaking. The preculture was used to inoculate 8^16 Erlenmeyer £asks containing 600 ml of the above medium and put on a rotary shaker at 130 rpm at RT. After 5 days, laccase expression was induced by adding 2,5-xylidine (0.2 mM). Laccase activity reached its maximum 24^48 h after induction. The mycelium was removed by ¢ltration and the medium was collected. The extracellular proteins were precipitated with ammonium sulphate (95% w/v saturation). The precipitant was extensively dialysed against 20 mM sodium phosphate, pH 7, at 4³C. Laccase was puri¢ed on a Whatman DE-53 anion exchange column equilibrated with the dialysis bu¡er and applying a linear salt gradient. Fractions were checked for laccase activity by oxidation of ABTS [14]. Laccase activity was found in two major peaks and in the £ow through (Fig. 1A). It has been shown by Otterbein and co-workers [15] that the monokaryotic strain SS3 from P. cinnabarinus produces two extracellular laccase isozymes (Lac I and Lac II). Large-scale fungal cultures were grown in a 15-l bio-reactor as described by Herpoe«l et al. [9] and used for laccase isolation and puri¢cation. A ¢ltered 10 day culture was concentrated on a 10 kDa Pellicon membrane and two successive ammonium sulphate precipitation steps (40% and 80% saturation (w/v)) were performed. The precipitated proteins were then dialysed against 25 mM sodium acetate bu¡er pH 5, loaded onto a DEAE^cellulose column equilibrated against dialysis bu¡er, and eluted with a linear gradient of 0^0.5 M NaCl. Fractions containing laccase activity were found in two major peaks (Fig. 1B), which were assigned to the two laccase isozymes described in [15], pooled and separately processed further. The purity of the laccase samples from T. versicolor and P. cinnabarinus was veri¢ed by sodium dodecyl sulphate^polyacrylamide gel electrophoresis (SDS^PAGE). The pure samples were then dialysed against 25 mM sodium acetate bu¡er pH 5, concentrated to about 10 mg/ml, and used for crystallisation experiments. Crystallisation experiments were performed applying the hanging-drop vapour di¡usion method with the sparse-matrix screening approach [16] using the commercially available crystal screen kits I+II from Hampton Research for initial trials. The conditions, which yielded crystalline material
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Fig. 1. Puri¢cation of laccases by anion exchange chromatography. (A) Elution pro¢le of TvL on a Whatman DE-53 column with a NaCl gradient (0^1.0 M). Fractions of peaks 1, 2, and FT contain laccase activity. (B) Elution pro¢le of P. cinnabarinus laccases on a Pharmacia Sepharose CL6B column with a NaCl gradient (0^0.5 M). Fractions of peaks 1 and 2 contain laccase activity and correspond to Lac I and Lac II, respectively.
were then further re¢ned. For the laccase from T. versicolor only the protein from the £ow through of the anion exchange column yielded crystals, while no crystals were obtained of protein from the two major peaks of laccase from P. cinnabarinus. Although all laccases migrated as single bands in a SDS^PAGE, corresponding to their expected molecular weight, we
suspected that the failure for crystallisation of the laccases of the other major bands was due to charge heterogeneity. In fact, an analytical iso-electric focusing (IEF) gel revealed the presence of several bands of peaks 1 and 2 of the two laccases (Figs. 1 and 2), while the protein of the £ow through of laccase from T. versicolor (TvL) gave a single band. In order to
Fig. 2. Separation of Lac I isoforms by IEF. (A) Shows a preparative IEF agarose gel (pI 2.5^5.0). The isozyme Lac I contains ¢ve isoforms (bands aP^d), which are visible by their typical blue colour. (B) An analytical, silver-stained IEF gel (5% polyacrylamide, pI 2.5^5.0) is depicted to show the charge homogeneity of the isolated Lac I isoforms (lanes aP^d). Lane Lac I is the original sample isolated by anion exchange chromatography (see Fig. 1B)
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Table 1 Conditions for laccase crystallisation Crystal
TvL_1
TvL_2
Lac Ic
Precipitant
20% polyethylen glycol 8000
Bu¡er Salt/additive
100 mM sodium cacodylate, pH 6.5 200 mM magnesium acetate
20% polyethylen glycol 8000/20% isopropanol 100 mM sodium citrate, pH 5.6 none
14.5% polyethylen glycol 8000/20% glycerol 100 mM sodium cacodylate, pH 5.9 160 mM zinc acetate, 5 mM copper sulphate
TvL_1 and TvL_2 designate the crystal form 1 and 2 of TvL, respectively. Lac Ic indicates the isoform c of Lac I. The remaining isoforms of Lac I (see Fig. 2) crystallise under very similar conditions as Lac Ic and are isomorphous with Lac Ic (data not shown).
obtain charge homogeneous Lac I and Lac II, we applied preparative IEF to these laccase isozymes. By this method ¢ve and seven isoforms from the two laccase isozymes from P. cinnabarinus could be isolated within a pI range of 3.4^3.8 for Lac I (Fig. 2) and a pI 6 2.5 for Lac II. Crystallisation conditions were established for all these isoforms. Only pure and charge homogeneous laccase could be successfully crystallised. In total, two crystal forms from TvL and one crystal form from each Lac I isoform were obtained (Table 1 and Fig. 3) which were suitable for high resolution crystal structure analysis. X-ray data were collected of the two TvL crystals and of crystals of one Lac I isoform using synchotron radiation (Table 1) and then processed with the HKL suite of programs [17]. The Lac II isoforms yielded single crystals too, but they were long and very thin needles, which could not be handled without being destroyed.
The only previous laccase crystal structure was determined as its Cu 2-depleted form, so it was necessary to verify that no copper loss had occurred from the laccases for which di¡raction data were collected. For this reason, crystals were dissolved in 0.1 M sodium acetate bu¡er pH 5.6, and the oxidation of ABTS monitored at 420 nm. These experiments proved that the crystals contained active enzyme, even after exposure to X-rays (Table 2). Initial molecular replacement experiments showed that the laccase structure from C. cinereus (PDB entry code 1a65) could serve as a good model for the structure solution of the two crystal forms from T. versicolor but not for the P. cinnabarinus laccase. This can be rationalised in terms of the primary structure similarities of the respective laccases. The laccase isozymes from T. versicolor have a sequence identity of about 70%, depending on the isozyme,
Fig. 3. Laccase crystals. (A) Shows TvL_1 crystals. These crystals have a longest dimension of about 1 mm. (B) depicts TvL_2 crystals, which grow in the presence of precipitated protein. They have a size of approximately 0.1U0.1U0.6 mm3 . (C) A close-up of a TvL_2 crystal showing a void in the upper part. Crystals with a void are usually larger, but the void does not a¡ect the di¡raction quality. (D) Crystals of Lac I, isoform c. These crystals are hexagonal plates with a size of about 0.5U0.4U0.15 mm3 and are always intergrown, which causes problems in isolating single crystals.
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Table 2 Crystal characteristics, data collection and processing statistics of laccase crystals Crystal
TvL_1
TvL_2
Lac Ic
Space group Cell dimensions î) a (A î) b (A î) c (A L (³) Number of molecules/a.u. X-ray source î) Wavelength (A T (K) Number of crystals î) Resolution (A Total number of re£ections Number of unique re£ections Completeness (%) I/c Rmerge (%)a
P21 21 21
P21 21 21
C2
73.9 83.4 101.5
83.6 85.0 91.5
1 BM01A at ESRF 0.880 100 2 23.0^2.95 180994 14031 68.6 16.6 7.6
1 BW7B at DESY 0.847 293 1 20.0^1.90 398559 52154 99.4 15.1 6.3
142.1 62.9 92.9 129.8 1 BM01A at ESRF 0.873 100 1 40.0^2.20 424035 27954 86.9 17.4 4.0
a
Rmerge : 4h 4i MI(h,i)3GI(h)fM/4h 4i I(h,i), where I(h,i) is the intensity of the ith measurement of re£ection h and GI(h)f is the mean value of I(h) for all i measurements.
when compared to the laccase from C. cinereus, but this value drops to about 55% for the comparison of P. cinnabarinus laccase isozymes with the C. cinereus laccase, (assuming the sequence of the dikaryotic strain [15]). P. cinnabarinus laccases do, however, have about 78% identical amino acids when compared to T. versicolor laccases, making the T. versicolor laccase structure then a good model for the structure solution of P. cinnabarinus laccase. The structure solutions will also be attempted using heavy atom methods such as multiple isomorphous replacement or multiple-wavelength anomalous dispersion, which takes advantage of the anomalous signal from the four copper ions. Micro-heterogeneity in laccases has been observed previously [18^20] and might be the reason for the failure of previous attempts to obtain crystals suitable for high resolution structure determination. There may be multiple causes of charge heterogeneity in proteins, e.g. the de-amidation of labile amidecontaining amino acids [21] and variations in the degree or nature of glycosylation [22]. Enzymatic deglycosylation of laccase was performed in an attempt to improve the di¡raction quality of single crystals [23], a procedure which apparently led to loss of the type-2 Cu [10]. To avoid this problem, we attempted the isolation of laccase isoforms by ion exchange
chromatography and preparative IEF methods. With protein of apparent charge homogeneity in hand, well di¡racting laccase crystals were obtained, allowing the high resolution structure analysis of functional and fully glycosylated laccases. Acknowledgements We gratefully acknowledge the opportunity to collect di¡raction data at the EMBL Outstation (DESY/Hamburg) and at the Swiss Norwegian Beamline (ESRF/Grenoble). This project was ¢nanced in part by a Grant of the SNF (Swiss National Science Foundation, No. 31-55681.98) to K.P. and by Grants from the BBW (Swiss Ministry of Education and Science, BBW Nr. 99.0585) to K.P. and from the Commission of the European Communities to M.A., within a project of the 5th European Framework Programme (PELAS QLK 3 1999 590).
References [1] F. Xu, Oxidation of phenols anilines, and benzenethiols by fungal laccases correlation between activity and redox poten-
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M. Antorini et al. / Biochimica et Biophysica Acta 1594 (2002) 109^114 tials as well as halide inhibition, Biochemistry 35 (1996) 7608^7614. F. Xu, W. Shin, S.H. Brown, J.A. Wahleithner, U.M. Sundaram, E.I. Solomon, A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit signi¢cant di¡erences in redox potential, substrate speci¢city, and stability, [erratum in Biochim. Biophys. Acta 1341 (1996) 99], Biochim. Biophys. Acta 1292 (1996) 303^311. C.F. Thurston, The structure and function of fungal laccases, Microbiology 140 (1994) 19^26. A. Messerschmidt, Spatial structure of ascorbate oxidase, laccase and related proteins: implications for the catalytic mechanism, in: A. Messerschmidt (Ed.), Multi-Copper Oxidases, Singapore, 1997, pp. 23^79. H. Yoshida, Chemistry of Lacquer (Urushi) part 1, J. Chem. Soc. Tokyo 43 (1883) 472^486. N.G. Lewis, L.B. Davin, S. Sarkanen, in: Lewis, Sarkanen (Eds.), Lignin and Lignan Biosynthesis, ACS Symposium Series 697, American Chemical Society, Washington, DC, 1998. C.S. Evans, Laccase activity in lignin degradation by Coriolus versicolor, FEMS Microbiol. Lett. 27 (1985) 339^343. C. Johannes, A. Majcherczyk, Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems, Appl. Environ. Microbiol. 66 (2000) 524^ 528. I. Herpoe«l, H. Jeller, G. Fang, M. Petit-Conil, R. Bourbonnais, J.-L. Robert, M. Asther, J.-C. Sigoillot, E¤cient enzymatic deligni¢cation of wheat straw pulp by a sequential xylanase-laccase mediator treatment, J. Pulp Paper Sci. (2002) in press. V. Ducros, A.M. Brzozowski, K.S. Wilson, S.H. Brown, P. Òstergaard, P. Schneider, D.S. Yaver, A.H. Pedersen, G.J. Davies, Crystal structure of the type-2 Cu depleted laccase from Coprinus cinereus at 2.2 A resolution, Nat. Struct. Biol. 5 (1998) 310^316. H.P. Call, I. Mu«cke, History, overview and applications of mediated lignolytic systems, especially laccase-mediator-systems (Lignozym0 -process), J. Biotechnol. 53 (1997) 163^202. L. Otterbein, E. Record, S. Longhi, M. Asther, S. Moukha, Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris, Eur. J. Biochem. 267 (2000) 1619^1625.
[13] G. Fahraeus, B. Reinhammar, Large scale production and puri¢cation of laccase from cultures of the fungus Polyporus versicolor and some properties of laccase A, Acta Chem. Scand. 21 (1967) 2367^2378. [14] B.S. Wolfenden, R.L. Wilson, Radical-cations as reference chromogens in kinetic studies of one-electron transfer reactions, J. Chem. Soc. Perkin Trans. II (1982) 805^812. [15] L. Otterbein, E. Record, D. Chereau, I. Herpoel, M. Asther, S.M. Moukha, Isolation of a new laccase isoform from the white-rot fungi Pycnoporus cinnabarinus strain ss3, Can. J. Microbiol. 46 (2000) 759^763. [16] J. Jankaric, S.-H. Kim, Sparse matrix sampling: a screening method for crystallization of proteins, J. Appl. Crystallogr. 24 (1991) 409^411. [17] Z. Otwinowski, W. Minor, Processing of X-ray di¡raction data collected in oscillation mode, Methods Enzymol. 276 (1997) 307^326. [18] K. Esser, W. Minuth, The phenoloxidases of the ascomycete Podospora anserina. Microheterogeneity of laccase II, Eur. J. Biochem. 23 (1971) 484^488. [19] C. Eggert, U. Temp, K.-E.L. Eriksson, The ligninolytic sytem of the white rot fungus Pycnoporus cinnabarinus. Puri¢cation and characterization of the laccase, Appl. Environ. Microbiol. 62 (1996) 1151^1158. [20] A.M. Farnet, S. Criquet, S. Tagger, G. Gil, J. Le Petit, Puri¢cation, partial characterization, and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17, Can. J. Microbiol. 46 (2000) 189^194. [21] T. Solstad, T. Flatmark, Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids. E¡ects on catalytic and stability properties, Eur. J. Biochem. 267 (2000) 6302^6310. [22] D. Macmillan, R.M. Bill, K.A. Sage, D. Fern, S.L. Flitsch, Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin, Chem. Biol. 8 (2001) 133^145. [23] V. Ducros, G.J. Davies, D.M. Lawson, K.S. Wilson, S.H. Brown, P. Òstergaard, A.H. Pedersen, P. Schneider, D.S. Yaver, A.M. Brzozowski, Crystallization and preliminary X-ray analysis of the laccase from Coprinus cinereus, Acta Crystallogr. D53 (1997) 605^607.
BBAPRO 36517 24-1-02 Cyaan Magenta Geel Zwart
Puri¢cation, crystallisation and X-ray di¡raction study of fully functional laccases from two ligninolytic fungi Matteo Antorini a , Isabelle Herpoe«l-Gimbert a , Thomas Choinowski a , JeanClaude Sigoillot b , Marcel Asther b , Kaspar Winterhalter a , Klaus Piontek a; * b
a Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Universita«tstrasse 16, CH-8092 Zu«rich, Switzerland Unite¨ INRA de Biotechnologie des Champignons Filamenteux, INRA, IFR 86 BAIM, Universite¨s de Provence et de la Me¨diterrane¨e, Faculte¨ des Sciences de Luminy, ESIL, 163 Avenue de Luminy, Case Postale 925, F-13288 Marseille Cedex, France
Received 18 July 2001; accepted 21 September 2001
Abstract Laccase isozymes from the white-rot basidiomycete fungi Trametes versicolor and Pycnoporus cinnabarinus were purified to apparent iso-electric homogeneity and crystallised. T. versicolor laccase crystallises in two crystal forms, both with the î resolution, respectively. The crystals of P. cinnabarinus orthorhombic space group P21 21 21 , which diffract to 1.9 and 2.95 A î resolution. All the laccase crystals are suitable laccase belong to the monoclinic space group C2 and diffract to at least 2.2 A for X-ray structure determination and contain a full complement of copper ions. ß 2002 Elsevier Science B.V. All rights reserved. Keywords: Laccase; Micro-heterogeneity; Crystal; Structure; Fungus
Laccase (benzenediol oxygen oxidoreductase, EC 1.10.3.2), a polyphenol oxidase belongs to the family of blue multi-copper oxidases. Laccases oxidise a broad range of substrates such as polyphenols, methoxy-substituted phenols, diamines and even some inorganic compounds [1^3]. The one-electron oxidation of these reducing substrates occurs concomi-
Abbreviations: ABTS, 2,2P-azino-bis-[3-ethylthiazoline-6sulphonate] ; Lac I and Lac II, laccase isozymes I and II, respectively, from Pycnoporus cinnabarinus; IEF, iso-electric focusing; LiP, lignin peroxidase; MnP, manganese peroxidase; RT, room temperature; SDS^PAGE, sodium dodecyl sulphate^polyacrylamide gel electrophoresis ; TvL, laccase from Trametes versicolor * Corresponding author. Fax: +41-1-632-1121. E-mail address: [email protected] (K. Piontek).
tantly with a four-electron reduction of molecular oxygen to water (for a review see [4]). The laccase enzyme, which was ¢rst discovered more than one century ago in the Japanese tree Rhus venicifera [5], is widely distributed among plants, where it is implicated in wound-response and the synthesis of lignin, the complex polymer that constitutes a main component of the plant cell wall [6]. In contrast, the laccases produced by some wood-rotting fungi from the genus basidiomycete play an important role in the degradation of lignin. The laccases, and two peroxidases produced by the ligninolytic system of some white-rot fungi, lignin peroxidase (LiP) and manganese peroxidase (MnP) are the extracellular enzymes, which can substantially depolymerise lignin [7]. While LiP cleaves the nonphenolic aromatic bonds of lignin, MnP and the fun-
0167-4838 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 1 ) 0 0 2 8 9 - 8
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gal laccases can oxidise only the phenolic components of lignin. In the presence of synthetically produced mediators such as 2,2P-azino-bis-[3-ethylthiazoline-6-sulphonate] (ABTS), the oxidising capability of the laccases is expanded to include the oxidation of non-phenolic compounds [8], and unlike the ligninolytic enzymes LiP and MnP, which require hydrogen peroxide for activation, the laccases are oxidised by O2 . The possibility to extend the substrate range of laccases in the presence of speci¢c mediators and the ability of some fungal laccases to degrade recalcitrant aromatic compounds with high redox potentials, gives this class of enzymes great potential value to the development of environmentally safe processes in the paper and pulp industries [9] as well as in the bioremediation of soils and water. An obstacle that still has to be overcome is that the mechanism by which the laccases attain their speci¢c redox potential is only poorly understood [1,2]. A three-dimensional structure of this enzyme may help to clarify this issue. Despite the many e¡orts to crystallise laccase and the fact that it has been under investigation for many decades, a crystal structure of laccase was reported only recently [10]. Unfortunately, this Coprinus cinereus laccase is of a Cu-depleted form, in which the putative type-2 Cu is completely absent. Trametes versicolor and Pycnoporus cinnabarinus are typical white-rot fungi that have been extensively investigated due to their ligninolytic capability [11]. The P. cinnabarinus laccase gene has been cloned and expressed in Pichia pastoris [12] and more recently in Aspergillus niger. The active recombinant form of laccase was expressed, opening the way for site-directed mutagenesis. This, in combination with structural studies, will give us more insight into the functionality of this enzyme. We report the ¢rst successful crystallisation of fully active and fully glycosylated laccases containing a full complement of coppers. Precultures of T. versicolor (ATCC 20869) were obtained by inoculating mycelial fragments into a glass Erlenmeyer £ask containing 250 ml of a medium prepared according to Fahreus and Reinhammar [13]. Several glass marbles were added to disrupt the mycelium. The culture medium was incubated at room temperature (RT) on a rotary shaker for 0.5 h
and subsequently allowed to grow for 24 h without shaking. The preculture was used to inoculate 8^16 Erlenmeyer £asks containing 600 ml of the above medium and put on a rotary shaker at 130 rpm at RT. After 5 days, laccase expression was induced by adding 2,5-xylidine (0.2 mM). Laccase activity reached its maximum 24^48 h after induction. The mycelium was removed by ¢ltration and the medium was collected. The extracellular proteins were precipitated with ammonium sulphate (95% w/v saturation). The precipitant was extensively dialysed against 20 mM sodium phosphate, pH 7, at 4³C. Laccase was puri¢ed on a Whatman DE-53 anion exchange column equilibrated with the dialysis bu¡er and applying a linear salt gradient. Fractions were checked for laccase activity by oxidation of ABTS [14]. Laccase activity was found in two major peaks and in the £ow through (Fig. 1A). It has been shown by Otterbein and co-workers [15] that the monokaryotic strain SS3 from P. cinnabarinus produces two extracellular laccase isozymes (Lac I and Lac II). Large-scale fungal cultures were grown in a 15-l bio-reactor as described by Herpoe«l et al. [9] and used for laccase isolation and puri¢cation. A ¢ltered 10 day culture was concentrated on a 10 kDa Pellicon membrane and two successive ammonium sulphate precipitation steps (40% and 80% saturation (w/v)) were performed. The precipitated proteins were then dialysed against 25 mM sodium acetate bu¡er pH 5, loaded onto a DEAE^cellulose column equilibrated against dialysis bu¡er, and eluted with a linear gradient of 0^0.5 M NaCl. Fractions containing laccase activity were found in two major peaks (Fig. 1B), which were assigned to the two laccase isozymes described in [15], pooled and separately processed further. The purity of the laccase samples from T. versicolor and P. cinnabarinus was veri¢ed by sodium dodecyl sulphate^polyacrylamide gel electrophoresis (SDS^PAGE). The pure samples were then dialysed against 25 mM sodium acetate bu¡er pH 5, concentrated to about 10 mg/ml, and used for crystallisation experiments. Crystallisation experiments were performed applying the hanging-drop vapour di¡usion method with the sparse-matrix screening approach [16] using the commercially available crystal screen kits I+II from Hampton Research for initial trials. The conditions, which yielded crystalline material
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Fig. 1. Puri¢cation of laccases by anion exchange chromatography. (A) Elution pro¢le of TvL on a Whatman DE-53 column with a NaCl gradient (0^1.0 M). Fractions of peaks 1, 2, and FT contain laccase activity. (B) Elution pro¢le of P. cinnabarinus laccases on a Pharmacia Sepharose CL6B column with a NaCl gradient (0^0.5 M). Fractions of peaks 1 and 2 contain laccase activity and correspond to Lac I and Lac II, respectively.
were then further re¢ned. For the laccase from T. versicolor only the protein from the £ow through of the anion exchange column yielded crystals, while no crystals were obtained of protein from the two major peaks of laccase from P. cinnabarinus. Although all laccases migrated as single bands in a SDS^PAGE, corresponding to their expected molecular weight, we
suspected that the failure for crystallisation of the laccases of the other major bands was due to charge heterogeneity. In fact, an analytical iso-electric focusing (IEF) gel revealed the presence of several bands of peaks 1 and 2 of the two laccases (Figs. 1 and 2), while the protein of the £ow through of laccase from T. versicolor (TvL) gave a single band. In order to
Fig. 2. Separation of Lac I isoforms by IEF. (A) Shows a preparative IEF agarose gel (pI 2.5^5.0). The isozyme Lac I contains ¢ve isoforms (bands aP^d), which are visible by their typical blue colour. (B) An analytical, silver-stained IEF gel (5% polyacrylamide, pI 2.5^5.0) is depicted to show the charge homogeneity of the isolated Lac I isoforms (lanes aP^d). Lane Lac I is the original sample isolated by anion exchange chromatography (see Fig. 1B)
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Table 1 Conditions for laccase crystallisation Crystal
TvL_1
TvL_2
Lac Ic
Precipitant
20% polyethylen glycol 8000
Bu¡er Salt/additive
100 mM sodium cacodylate, pH 6.5 200 mM magnesium acetate
20% polyethylen glycol 8000/20% isopropanol 100 mM sodium citrate, pH 5.6 none
14.5% polyethylen glycol 8000/20% glycerol 100 mM sodium cacodylate, pH 5.9 160 mM zinc acetate, 5 mM copper sulphate
TvL_1 and TvL_2 designate the crystal form 1 and 2 of TvL, respectively. Lac Ic indicates the isoform c of Lac I. The remaining isoforms of Lac I (see Fig. 2) crystallise under very similar conditions as Lac Ic and are isomorphous with Lac Ic (data not shown).
obtain charge homogeneous Lac I and Lac II, we applied preparative IEF to these laccase isozymes. By this method ¢ve and seven isoforms from the two laccase isozymes from P. cinnabarinus could be isolated within a pI range of 3.4^3.8 for Lac I (Fig. 2) and a pI 6 2.5 for Lac II. Crystallisation conditions were established for all these isoforms. Only pure and charge homogeneous laccase could be successfully crystallised. In total, two crystal forms from TvL and one crystal form from each Lac I isoform were obtained (Table 1 and Fig. 3) which were suitable for high resolution crystal structure analysis. X-ray data were collected of the two TvL crystals and of crystals of one Lac I isoform using synchotron radiation (Table 1) and then processed with the HKL suite of programs [17]. The Lac II isoforms yielded single crystals too, but they were long and very thin needles, which could not be handled without being destroyed.
The only previous laccase crystal structure was determined as its Cu 2-depleted form, so it was necessary to verify that no copper loss had occurred from the laccases for which di¡raction data were collected. For this reason, crystals were dissolved in 0.1 M sodium acetate bu¡er pH 5.6, and the oxidation of ABTS monitored at 420 nm. These experiments proved that the crystals contained active enzyme, even after exposure to X-rays (Table 2). Initial molecular replacement experiments showed that the laccase structure from C. cinereus (PDB entry code 1a65) could serve as a good model for the structure solution of the two crystal forms from T. versicolor but not for the P. cinnabarinus laccase. This can be rationalised in terms of the primary structure similarities of the respective laccases. The laccase isozymes from T. versicolor have a sequence identity of about 70%, depending on the isozyme,
Fig. 3. Laccase crystals. (A) Shows TvL_1 crystals. These crystals have a longest dimension of about 1 mm. (B) depicts TvL_2 crystals, which grow in the presence of precipitated protein. They have a size of approximately 0.1U0.1U0.6 mm3 . (C) A close-up of a TvL_2 crystal showing a void in the upper part. Crystals with a void are usually larger, but the void does not a¡ect the di¡raction quality. (D) Crystals of Lac I, isoform c. These crystals are hexagonal plates with a size of about 0.5U0.4U0.15 mm3 and are always intergrown, which causes problems in isolating single crystals.
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Table 2 Crystal characteristics, data collection and processing statistics of laccase crystals Crystal
TvL_1
TvL_2
Lac Ic
Space group Cell dimensions î) a (A î) b (A î) c (A L (³) Number of molecules/a.u. X-ray source î) Wavelength (A T (K) Number of crystals î) Resolution (A Total number of re£ections Number of unique re£ections Completeness (%) I/c Rmerge (%)a
P21 21 21
P21 21 21
C2
73.9 83.4 101.5
83.6 85.0 91.5
1 BM01A at ESRF 0.880 100 2 23.0^2.95 180994 14031 68.6 16.6 7.6
1 BW7B at DESY 0.847 293 1 20.0^1.90 398559 52154 99.4 15.1 6.3
142.1 62.9 92.9 129.8 1 BM01A at ESRF 0.873 100 1 40.0^2.20 424035 27954 86.9 17.4 4.0
a
Rmerge : 4h 4i MI(h,i)3GI(h)fM/4h 4i I(h,i), where I(h,i) is the intensity of the ith measurement of re£ection h and GI(h)f is the mean value of I(h) for all i measurements.
when compared to the laccase from C. cinereus, but this value drops to about 55% for the comparison of P. cinnabarinus laccase isozymes with the C. cinereus laccase, (assuming the sequence of the dikaryotic strain [15]). P. cinnabarinus laccases do, however, have about 78% identical amino acids when compared to T. versicolor laccases, making the T. versicolor laccase structure then a good model for the structure solution of P. cinnabarinus laccase. The structure solutions will also be attempted using heavy atom methods such as multiple isomorphous replacement or multiple-wavelength anomalous dispersion, which takes advantage of the anomalous signal from the four copper ions. Micro-heterogeneity in laccases has been observed previously [18^20] and might be the reason for the failure of previous attempts to obtain crystals suitable for high resolution structure determination. There may be multiple causes of charge heterogeneity in proteins, e.g. the de-amidation of labile amidecontaining amino acids [21] and variations in the degree or nature of glycosylation [22]. Enzymatic deglycosylation of laccase was performed in an attempt to improve the di¡raction quality of single crystals [23], a procedure which apparently led to loss of the type-2 Cu [10]. To avoid this problem, we attempted the isolation of laccase isoforms by ion exchange
chromatography and preparative IEF methods. With protein of apparent charge homogeneity in hand, well di¡racting laccase crystals were obtained, allowing the high resolution structure analysis of functional and fully glycosylated laccases. Acknowledgements We gratefully acknowledge the opportunity to collect di¡raction data at the EMBL Outstation (DESY/Hamburg) and at the Swiss Norwegian Beamline (ESRF/Grenoble). This project was ¢nanced in part by a Grant of the SNF (Swiss National Science Foundation, No. 31-55681.98) to K.P. and by Grants from the BBW (Swiss Ministry of Education and Science, BBW Nr. 99.0585) to K.P. and from the Commission of the European Communities to M.A., within a project of the 5th European Framework Programme (PELAS QLK 3 1999 590).
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