Atypical laccase isoenzymes from copper supplemented Pleurotus ostreatus cultures

Enzyme and Microbial Technology 33 (2003) 220–230

Atypical laccase isoenzymes from copper supplementedPleurotus ostreatuscultures�

Gianna Palmieria,c, Giovanna Cennamob, Vincenza Faracob,Angela Amoresanob,c, Giovanni Sanniab, Paola Giardinab,c,∗

a ISPAAM, Consiglio Nazionale delle Ricerche, via Argine 1085, 80147 Napoli, Italyb Dipartimento di Chimica Organica e Biochimica, Università di Napoli “Federico II”,

Complesso Universitario Monte S. Angelo, via Cinthia, 80126 Napoli, Italyc Centro Regionale di Competenza Applicazioni Tecnologico-Industriali Di Biomolecole e Biosistemi, Regione Campania, Italy

Received 22 February 2003; accepted 26 April 2003

Abstract

Two strictly related laccase isoenzymes (POXA3a and POXA3b), produced byPleurotus ostreatusin copper supplemented cultures,have been purified and characterised. Both the native proteins were found to be constituted by a large subunit (67 kDa) and a small subunit(18 or 16 kDa). Peptide mapping of the 18 and 16 kDa polypeptides from POXA3a and POXA3b suggests the identity of the 18 kDasubunits and the generation of the 16 kDa polypeptides from the 18 kDa ones. Structural data on POXA3a and POXA3b do not allowascertaining significant differences between the two isoenzymes. On the other hand, dissociation of POXA3a complex is observed in 3 Murea, whilst POXA3b complex is not dissociated even in 6 M urea. Evidences are reported on the role played by extracellular proteases inthe activation of these isoenzymes. The sequence of a unique gene and of the corresponding cDNA, encoding the 67 kDa POXA3 subunit,has been determined.© 2003 Elsevier Science Inc. All rights reserved.

Keywords:Fungi; Phenol oxidase; Glycoside moiety; Subunit

1. Introduction

White rot fungi produce various isoforms of ligninolyticenzymes (lignin peroxidases, laccases, manganese peroxi-dases) which catalyse one-electron oxidation of lignin unitsproducing radicals[1,2]. These enzymes oxidise a broadspectrum of structurally different substrates such as highlytoxic phenolic compounds and azo dyes. Recently, increasedattention has been focused on laccases because of their pos-sible applications in fields such as pulping, textile dyes, pol-luted water detoxification and many others[3,4].

Laccases are phenol oxidases, which reduce oxygen towater and simultaneously perform a one electron oxida-tion of many aromatic substrates[5]. The substrate rangeof these enzymes can be extended to include non-phenoliclignin subunits in the presence of readily oxidisable primary

� Nucleotide sequence accession number: the sequence of POXA3 laccasegene reported in this paper has been entered in the EMBL Data Libraryand assigned accession number AJ344434.

∗ Corresponding author. Tel.:+39-081-674319; fax:+39-071-674313.E-mail address:[email protected] (P. Giardina).

substrates, which can act as electron-transfer mediators[6].Laccases belong to the group of blue copper oxidases andcontain four copper atoms/molecule, distributed in three dif-ferent copper-binding sites[7]. Structure and organisationof laccase copper-binding sites is shared by other multicop-per proteins, which have physiological roles not apparentlyrelated to phenol oxidase activity (copper tolerance, irontransport and sporulation)[8]. As a fact, the overall struc-ture of CueO, a protein involved inEscherichia colicop-per tolerance, is similar to that of laccases and the proteinis able to oxidise a wide variety of substrates, including2,6-dimethoxyphenol (DMP)[9]. Therefore, nowadays theboundary between enzymatic and non-enzymatic blue mul-ticopper proteins is ever more unclear.

It has been reported that laccases are secreted in multipleisoforms depending on the fungal species and environmen-tal conditions, but whether this multiplicity corresponds toa real different functional role of the various isoenzymes isstill unclear[10–13]. The biochemical diversity of laccaseisoenzymes appears to be due to the multiplicity of laccasegenes; however, regulation of their expression can be sub-stantially diverse between fungal species.

0141-0229/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved.doi:10.1016/S0141-0229(03)00117-0

G. Palmieri et al. / Enzyme and Microbial Technology 33 (2003) 220–230 221

Laccase isoenzymes produced byPleurotus ostreatus, awhite rot basidiomycete fungus, have been extensively stud-ied. One of these POXC is the most abundantly producedunder all the growth conditions examined[14]. Three otherisoenzymes secreted by the mycelium have also been pu-rified and characterised (POXA1a, POXA1b and POXA2)[15,16].

Studies on the laccase coding genes inP. ostreatushaveso far led to identification of three different genes and of thecorresponding cDNAs,poxc(previously namedpox2), pox1(which codes for a laccase isoenzyme that has not been iden-tified yet) andpoxa1b[14,16,17]. Furthermore, it has beendemonstrated that, in copper-supplemented cultures, theamount of all laccase isoenzymes increases substantially andin these conditions laccase expression is regulated at tran-scriptional level[18]. A similar behaviour has been noticedfor other fungal laccases[10,19]as well as, more obviously,for protein involved in copper tolerance (i.e. CueO)[20].

Recently, it has been reported thatP. ostreatusextracel-lular proteases can play a regulatory role in laccase activity[21]. As a fact, POXA1b isoenzyme is degraded when fil-tered culture broth, treated with phenylmethylsulfonyl fluo-ride (PMSF), is incubated with the subtilisin-likePoSl, aP.ostreatusextracellular protease[21].

This paper reports the characterisation of two strictlyrelated P. ostreatus laccase isoenzymes (POXA3a andPOXA3b) and the effect of extracellular proteases on them.Furthermore, sequence ofpoxa3gene and of the correspond-ing cDNA has been determined and the deduced aminoacid sequence has been verified against both POXA3a andPOXA3b, by means of matrix-assisted laser desorptionionisation (MALDI)-MS analysis.

2. Materials and methods

2.1. Organism and culture conditions

White-rot fungus,P. ostreatus(Jacq.:Fr.) Kummer (type:Florida) (ATCC no. MYA-2306) was maintained throughperiodic transfer at 4◦C on potato dextrose agar plates (DifcoLaboratories, Detroit, MI) in the presence of 0.5% yeastextract (Difco).

Incubations were carried out as previously described[15]. Fifty millilitres of a 5-day-old culture were trans-ferred in 1 l flasks containing 450 ml broth supplementedwith 150�M CuSO4. Enzyme purification was performedfrom a 10 days culture. Fungal culture in the presence of aprotease inhibitor was performed by adding 0.1 mM PMSFafter 2 days of growth. The broth was filtered 24 h after theaddition of PMSF.

2.2. Enzyme purification

Secreted proteins were precipitated from the filteredmedium by addition of (NH4)2SO4 up to 80% saturation

and, after extensive dialysis, loaded onto a DEAE SepharoseFast Flow (Pharmacia Biotech Inc.) column as previouslydescribed[15]. Two fractions containing laccase activity,recovered with the equilibrating buffer, were separatelypooled, concentrated on an Amicon PM-10 membrane andequilibrated in Tris–HCl 50 mM pH 8.0. Each pool wasloaded onto an anion exchange Mono Q HR5/5 (Pharma-cia) column in a fast protein liquid chromatography sys-tem (FPLC, Pharmacia) equilibrated with the same buffer.The enzyme was eluted with a linear gradient (buffer B:Tris–HCl 50 mM, pH 8.0, 0.3 M NaCl; gradient:t = 0 min,%B = 0; t = 10 min, %B = 0; t = 70 min, %B = 60;t = 75 min, %B= 80). The active fractions were pooled,concentrated and loaded onto a gel filtration Superdex 75PC 3.2/30 column in a SMART System (Pharmacia). Thecolumn was eluted with 50 mM sodium phosphate buffer(pH 7.0), containing 150 mM NaCl (flow rate: 0.05 ml/min);the active fractions were pooled and desalted.

The same purification procedure was used to purify lac-case isoenzymes from PMSF amended cultures.

2.3. Enzyme assays

Laccase activity was assayed at 25◦C, using 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), DMPand syringaldazine as substrates as previously described[15]. Activity as function of pH was measured using a McIlvaine’s citrate–phosphate buffer adjusted to different pHvalues in the range 2.5–7.5. The same buffer was used to de-termine the stability at pH 3.0, 5.0, 7.0 and 8.0. Activity wasmeasured using ABTS as substrate unless otherwise stated.

2.4. Protein determination

Protein concentration was determined using the BioRadProtein Assay (BioRad), using bovine serum albumin asstandard.

2.5. Molecular mass determination

Molecular mass of native phenol oxidases was determinedby gel filtration chromatography on a SMART system (Phar-macia) using a Superdex 75 PC 3.2/30 column. The columnwas eluted with 50 mM sodium phosphate buffer (pH 7.0),containing 150 mM NaCl. Calibration was performed withbovine serum albumin (66 kDa), ovalbumin (45 kDa), car-bonic anhydrase (29 kDa) and cytochrome C (12.4 kDa) asstandards.

2.6. Electrophoresis and isoelectrofocusing

Polyacrylamide (12%) gel slab electrophoresis in 0.1%SDS was carried out as described by Laemmli[22].For molecular mass determination the gel was calibratedwith �-galactosidase (116.0 kDa), bovine serum albumin

222 G. Palmieri et al. / Enzyme and Microbial Technology 33 (2003) 220–230

(66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase(35 kDa), restriction endonucleaseBsp98l (25.0 kDa),�-lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa). An-alytical isoelectrofocusing in the pH range 2.5–7.0 wasperformed on 5.0% acrylamide slab gel using LKB Mul-tiphor electrophoresis system (Pharmacia) following themanufacturer’s instructions.

Native PAGE was performed at alkaline pH undernon-denaturing conditions. Separating and stacking gelswere at 9 and 4% acrylamide, respectively; buffer solu-tions were: 50 mM Tris–HCl, pH 9.5 for separating gel and18 mM Tris–HCl, pH 7.5 for stacking gel; the electrodereservoir solution was 25 mM Tris, 190 mM glycine, pH8.4. Gels were stained using Coomassie or laccase activityprocedure using ABTS as substrate.

2.7. Western blots

Proteins were separated on SDS–PAGE and elec-troblotted onto ProBlottTM Membrane (Applied Biosys-tems). Electroblotting was performed in 10 mM CAPS(3-(cyclohexylamino)-1-propanesulfonic acid), pH 11.0,10% (v/v) methanol at 50 V for 180 min at room tem-perature. Washing solution was 5% (w/v) dried milk inphosphate-buffered saline solution supplemented with0.2% (v/v) Triton X-100 (washing buffer). The membranewas washed and incubated with anti-POXA1b antibodies(cross-reacting with POXA3) diluted 1:100 in washingbuffer, at room temperature for 1 h, under continuous shak-ing. Subsequently, the membrane was washed and incubatedas above described with anti-rabbit IgG, peroxidase conju-gate (Sigma Chemical Co., St. Louis, MO), diluted 1:2000in washing buffer. Blots were visualised with 100 mMTris–HCl, pH 7.5, 0.5 mg/ml 3,3′-diaminobenzidine, 0.03%(w/v) NiCl2, 0.006% (v/v) H2O2.

2.8. Lectin assay

Protein samples and control standard glycoproteins (sup-plied with the Boehringer glycan differentiation kit) (1�g)were directly spotted onto an immobilon membrane and im-munologically detected after binding to lectins conjugatedwith digoxigenin following the manufacturer’s instructions(Boehringer Manneheim). Proteins linked to lectins weredetected by colorimetric reaction. Immunological detectionwas performed according to the manufacturer’s instructions.All experiments have been performed in duplicate on twodifferent enzymatic preparations.

2.9. In situ digestion

Mass spectrometric analyses were peformed on theCoomassie blue-stained proteins excised from a prepar-ative SDS electrophoresis on a 12% polyacrylamide gel.Excised bands were washed with acetonitrile and then with

0.1 M ammonium bicarbonate. Protein samples were re-duced, by incubation in 10 mM dithiothreitol for 45 min at56◦C, and carboxamidomethylated by using 55 mM iodoac-etamide in 0.1 M NH4HCO3 for 30 min, in the dark, undernitrogen atmosphere at room temperature. The gel parti-cles were then washed with ammonium bicarbonate andacetonitrile.

Enzymatic digestions were carried out with trypsin orchymotrypsin (15 mg/ml) in 50 mM ammonium bicarbon-ate, pH 8.5 at 4◦C for 4 h. The buffer solution was thenremoved and a new aliquot of the enzyme/buffer solutionwas added for 18 h at 37◦C. A minimum reaction vol-ume, sufficient for complete rehydratation of the gel, wasused. Peptides were then extracted, washing the gel parti-cles with 20 mM ammonium bicarbonate and 0.1% trifluo-roacetic acid in 50% acetonitrile at room temperature andthen lyophilised. Aliquots of the digests were directly anal-ysed by MALDI-MS or separated on a narrow bore VydacC18 column (25 cm×0.21 cm, 5�m) (The Separation Group,Hesperia, CA) using 0.1% trifluoroacetic acid (Sigma) assolvent A and 0.07% trifluoroacetic acid in 95% acetoni-trile (Baker) as solvent B. A linear gradient of solvent Bfrom 5 to 65% in 60 min at flow rate of 0.2 ml/min was em-ployed. The UV absorbance of the eluent was monitoredat 220 nm.

2.10. Protein sequence analysis

Automated N-terminal degradation of electroblottedproteins or purified peptides was performed using aPerkin-Elmer Applied Biosystems 477A pulsed-liquid pro-tein sequencer equipped with a model 120A PTH-analyserfor the on-line identification and quantification of phenylthio-hydantoin-amino acids.

2.11. Mass spectrometry analysis

MALDI mass spectra were recorded using a VoyagerDE and Voyager DE Pro MALDI-TOF mass spectrome-ter (Applied Biosystems); a mixture of analyte solution,�-ciano-4-hydroxy-cinnamic acid or sinapinic acid as ma-trices, bovine insulin and horse heart myoglobin as stan-dards were applied to the sample plate and air-dried. Masscalibration was obtained using the quasi-molecular ions(MH+) from horse myoglobin (16,952.50m/z), bovine in-sulin (5734.59m/z) and �-ciano-4-hydroxy-cinnamic acid(379.06m/z) as internal standards. Raw data were analysedby using computer software provided by the manufac-turer and are reported as average masses or monoisotopicmasses.

2.12. Atomic absorption

Copper content was determined by atomic absorptionspectrometry using a Perkin-Elmer apparatus model 5100,equipped with Zeeman graphite furnace and autosampler.


2.13. Isolation and sequencing of poxa3 gene and cDNA

Amplification experiments ofP. ostreatusgenomic DNAwere performed at 60◦C annealing temperature, using thefollowing oligonucleotide couples: 5′-AAAACTGCAGA-TYAAYGTBGARCARGGBAA-3′ and 5′-AATCTAGACV-GCDGGVGCVCCYT-3′ (Y = T/C, R = G/A;V = G/C/A,B = G/T/C, D= G/A/T). A 500 bp fragment was obtained,cloned in pUC18 and sequenced. This fragment, labelled byrandom priming method, was used as probe to screen aP.ostreatusgenomic library[17]. Colony hybridisation exper-iments were carried out in 5×SSC at 65◦C (where 1×SSCis 0.15 M NaCl, 0.015 M sodium citrate).

Total RNA was extracted from lyophilised mycelia,harvested from 3 days culture, as described by Lucaset al. [23]. Reverse transcription reaction was performedusing Superscript II (Gibco BRL) and following themanufacturer’s instructions. Amplification experiments ofspecific cDNA were performed at 60◦C annealing tem-perature using the following oligonucleotide couple: 5′-ATGGTGCTCTCTACTAAGCTCGC-3′ and 5′-TTACTGG-AACTCGGGAGCGAGGCC-3′. The amplified fragmentwas cloned in the pUC18 plasmid and sequenced.

DNA preparation, sub-cloning and restriction analyseswere performed by standard methods according to Sambrooket al. [24]. Sequencing by the dideoxy chain-terminationmethod was performed by PRIMM Sequencing Service(Milan, Italy) using universal and specific oligonucleotideprimers.

3. Results

3.1. Purification of laccase isoenzymes POXA3a andPOXA3b

It has been previously reported that CuSO4 (up to150�M) induces laccase production inP. ostreatuscultures[18]. Copper supplemented culture broth was collected after10 days growth time and extracellular proteins were frac-tionated by DEAE Sepharose chromatography. Two activedifferent protein fractions (named POXA3a and POXA3b)were recovered with the equilibrating buffer, whilst the typ-ical peak corresponding to POXC isoenzyme was eluted at0.3 M NaCl. POXA3a and POXA3b were separately purifiedon Mono Q anionic chromatography at pH 8.0 and elutedat 60 mM NaCl and 90 mM NaCl, respectively by the salinegradient. A final purification step on gel filtration chro-matography was performed for both isoenzymes; POXA3aand POXA3b were recovered from gel filtration at the sameelution time. A summary of the purification procedure isshown inTable 1; the final specific activities of POXA3aand POXA3b were 2000 and 1050 U/mg, respectively.

When the purified isoenzymes were analysed bySDS–PAGE, a complex pattern consisting of three bands of67, 18 and 16 kDa was observed for both proteins (Fig. 1A).

Fig. 1. Coomassie stained SDS–PAGE (A) and native PAGE (B) of purifiedPOXA3a and POXA3b laccase isoenzymes.

No significant change in the electrophoretic pattern of thesesamples was observed even after several attempts to fur-ther purify the proteins. On the other hand, a single bandfor purified POXA3a and POXA3b was revealed either innative PAGE (Fig. 1B) or in isoelectrofocusing analyses.Isoelectric points of POXA3a and POXA3b are 4.1 and4.3 and the molecular mass, determined by gel filtrationchromatography, is 56 kDa for both proteins.

3.2. Metal content analyses

The absorption spectra of POXA3a and POXA3b dis-played the characteristic peak at 600 nm, due to the pres-ence of type I copper. The copper content was determinedby atomic absorption and the analyses showed a value of3.3 copper/protein (mol/mol) for both proteins.

3.3. Structural analyses

POXA3a and POXA3b samples were analysed forthe presence of oligosaccharides using the lectin-bindingassays. The proteins were specifically recognised byGalan-thus nivaliaagglutinin lectin, which binds to terminal man-nose residues, andDatura stramoniumagglutinin, specificfor galactose�(1–4)-N-acetyl-glucosamine. On the basis oflectin specificity, the presence of both high mannose andhybrid or complex type N-linked glycans is suggested.

The two isoenzymes were directly analysed by MALDI-MS producing the spectra shown inFig. 2. Three peakscould be identified in both spectra, the major componentexhibited a molecular mass centred at about 60,860.4 Dafor POXA3a (Fig. 2A) and 60,700.6 for POXA3b(Fig. 2B). The broadening of the signals might be very likelydue to the heterogeneity of the glycoforms. Moreover, themass spectra showed the occurrence of two other compo-nents whose molecular masses were measured as 16,870.5


Table 1Purification of POXA3a and POXA3b fromP. ostreatusculture broth

Purification step Total activity (U) Total proteins (mg) Specific activity (U/mg) Recovery (%)

Broth 30000 231 130 100NH4(SO4)2 precipitate 29220 61 479 97

DEAE SepharosePOXA3a 1800 7.8 231 6POXA3b 3400 5.9 580 11

Mono QPOXA3a 1150 0.9 1280 3.8POXA3b 2250 2.2 1020 7.5

Superdex 75POXA3a 800 0.4 2000 2.7POXA3b 1400 1.3 1050 4.7

and 18,103.8 Da for POXA3a (Fig. 2A) and 16,838.1 and18,090.1 Da for POXA3b (Fig. 2B). The N-terminal se-quences of the larger polypeptides are identical for POXA3aand POXA3b (first 21 amino acids,Fig. 3). Moreover,

Fig. 2. MALDI-MS spectra of purified POXA3a (A) and POXA3b (B) laccase isoenzymes.

the same analyses carried out on the four 16 and 18 kDapolypeptides revealed that all had a blocked N-terminus.

Aliquots of the two isoenzymes were separated bySDS–PAGE and 67, 18 and 16 kDa polypeptides from


Fig. 3. Amino acid sequence of POXA3 laccase fromP. ostreatus. The underlined stretches are: (—), sequences validated by MALDI-MS in POXA3a;( ), sequences validated by MALDI-MS in POXA3b; (- - -), sequences verified by direct analysis in POXA3a. Boxed regions have been used to designoligonucletide primers for amplification experiments.

POXA3a and POXA3b were reduced, alkylated with iodoac-etamide and digested with trypsin or chymotrypsin in situ.The peptides were extracted from the gel and the mix-tures were directly analysed by MALDI-MS. Mass spec-tral analyses of the larger subunits revealed no significantdifferences between peptide fingerprints of POXA3a andPOXA3b. Furthermore, nine tryptic peptides of the POXA3alarge subunit were sequenced after HPLC fractionation(dotted underlined sequences inFig. 3). All these peptideswere clearly homologous to reported sequences from otherknown fungal laccases. As far as the two small polypep-tides (16 and 18 kDa) is concerning, the same mass signalswere recorded in the spectra of peptide mixtures from bothPOXA3a and POXA3b, accounting for 60% of the 18 kDapolypeptide mass. Unfortunately, the MALDI-MS analy-ses did not allow mapping the whole sequences as wellas the difference between 18 and 16 kDa polypeptides, al-though the in situ digestion procedures were repeated twice.This was probably due to a not complete extraction of thepeptides from the gel or to the well-known MALDI sup-pression phenomena[25]. Five peptides from the POXA3a18 kDa component were fractionated by HPLC and thepurity of each fraction tested by MALDI-MS. These pep-tides fitted all the mass signals found in the MALDI-MSspectra described above and their sequences resulted to

be: TLPNSINLVR, QIHVNIPELNTLQASGGATDLTVR,QVNSLTTYTTMMSDLDPAIAAL, VMMLDAQQILTQ.Only one of the peptides (1932.5 Da) did not provide anysequence, therefore it should correspond to the blockedN-terminal peptide. None of the sequenced peptides showsignificant homology with proteins in data banks.

3.4. Catalytic properties

POXA3a and POXA3b are both able to oxidise ABTS,DMP and syringaldazine, but no significant activity againstguaiacol could be detected. No remarkable difference is ob-served between the two isoenzymes comparing their kineticparameters and optimum pH towards the above mentionedsubstrates (Table 2). Optimal temperature is about 35◦Cfor both proteins, using ABTS as substrate. POXA3a andPOXA3b are stable in the pH range 5.0–7.0 (t1/2 = 7 daysat pH 7.0 for both proteins,t1/2 = 6 and 10 days at pH5.0 for POXA3a and POXA3b, respectively), whilst the ac-tivity quickly decreases after incubation at pH 3.0 or 8.0.Thermal stability was studied for both isoenzymes at 40 and60◦C. Plots of the residual activity after incubation at 40◦Cversus time indicated at1/2 of 6 h for POXA3a and 14 hfor POXA3b. At 60◦C the activity of both isoenzymes washalved in few minutes.


Table 2POXA3a and POXA3b kinetic parameters

Substrate Optimum pH Km (mM) Kcat (min−1) Kcat/Km (mM min)−1

POXA3a POXA3b POXA3a POXA3b POXA3a POXA3b POXA3a POXA3b

ABTS 3.6 3.6 (7.0± 1) × 10−2 (7.4 ± 0.2) × 10−2 (4.4 ± 0.3) × 106 (9.5 ± 0.9) × 106 6.3 × 107 1.3 × 108

Syringaldazine 6.2 6.2 (3.6± 0.5) × 10−2 (7.9 ± 0.6) × 10−2 (1.7 ± 0.1) × 105 (7.0 ± 1) × 105 4.7 × 106 8.9 × 106

DMP 5.5 5.5 14± 2 8.8 ± 0.3 (1.4± 0.1) × 106 (1.2 ± 0.1) × 106 1.0 × 105 1.4 × 105

3.5. Cloning and sequencing of poxa3 gene and cDNA

On the basis of the sequence of POXA3a tryptic peptides(boxed peptides inFig. 3) oligonucleotide-primer mixtureswere designed and used in amplification experiments per-formed usingP. ostreatusgenomic DNA as template. The500 bp amplified fragment is homologous to known laccasegenes and encoded two sequenced POXA3a tryptic peptidesin addition to those used to design primers. AP. ostreatusgenomic library was screened using the amplified fragment.One of the positive clones analysed (clone 5) encompassedthe complete coding sequence shown inFig. 4, as well as110 bp upstream and 74 bp downstream. In order to obtaininformation on the 5′ and 3′ flanking regions, two DNA frag-ments covering about 500 bp at the 5′ and 3′ ends of clone5 were amplified and used as probes for further screening.Two clones (clone 13 and clone 3) were selected, analysedand sequenced. They overlap clone 5 for 1400 bp (clone 13)and 700 bp (clone 3), respectively. Clone 13 extends 1600 bpat 3′ non-coding region, whilst clone 3 extends 1300 bp at5′ non-coding region.

On the basis of the N-terminal sequence of the matureprotein, a signal peptide of 19 amino acids could be iden-tified. Two oligonucleotides were designed using the pre-dicted N and C terminus of the protein and were used toamplify the POXA3 encoding cDNA. The 1500 bp amplifiedfragment was cloned and sequenced, allowing the determi-nation of the whole structure of thepoxa3gene (Fig. 4). Allthe known peptide sequences from the 67 kDa polypeptidewere exactly found in the encoded sequence. On the otherhand, the analysis of the complete determined nucleotidesequence (5700 bp) revealed neither regions encoding thefour known peptide sequences from the 18 kDa polypeptide,nor sequences corresponding to genes codifying other lac-case isoenzymes. Furthermore, comparison of nucleotide se-quences coming from several positive clones, showed onlyfew differences, which do not determine any changes in theencoded protein sequence and are probably due to allelicvariants of the same gene, being theP. ostreatusstrain useda dikaryon.

The coding sequence is interrupted by 21 introns. Theother three knownP. ostreatuslaccase genes contain 19(pox1andpoxc[14,17]) and 15 (poxa1b[16]) introns. Com-parison among the fourPleurotuslaccase gene structures,performed by aligning the corresponding encoded aminoacids, shows that the position of only eight introns is con-

served. In the 5′-flanking region, shown inFig. 4, severalstretches have been identified that closely match consen-sus sequences of regulatory elements, such as metal re-sponsive element (MRE) and xenobiotic responsive element(XRE).

3.6. Validation of the primary structure of POXA3aand POXA3b and characterisation of theirglycoside moieties

MALDI mass analyses performed on both the intactisoenzymes showed that the major components at about60 kDa consist of a heterogeneous population of glycoforms.These molecular masses could be tentatively attributed toa polypeptide chain of about 55 kDa, in agreement withthe mass values (55,577.5 Da) estimated on the basis ofthe cDNA sequence, and a glycoside moiety accountingfor about 5000 Da. However, the measured mass valuesrepresent the average mass of the glycoproteins due to thecontribution of the various glycoforms and could not beused either to determine the real molecular mass of theenzymes or to define the structure of each oligosaccharidechain.

Analyses of trypsin and chymotrypsin mass mapping ofPOXA3a and POXA3b large subunits were performed. Sig-nals were assigned to the corresponding peptide within theamino acid sequence deduced from the unique cDNA se-quence available, on the basis of the mass values and pro-tease specificities. These data allowed the verification of 64and 63% of the POXA3a and POXA3b primary structure,respectively, as shown inFig. 3. It should be noted that thetwo mass-mapping fingerprints validate different regions ofthe deduced amino acid sequence.

As indicated in Table 3, in the high mass region ofthe trypsin POXA3a spectrum, a series of related sig-nals were recorded and assigned to the peptide 249–265containing Asn256 modified by high-mannose-type gly-cans ranging from 5 to 8 mannose residues. Similar re-sults were obtained for the POXA3b isoenzyme, wherethe occurrence of Hex9HexNAc2 (Hex, hexose; HexNAc,N-acetylhexosamine) glycan structure was also detected.Results indicate the presence of a similar distribution ofglycans also for the peptide 422–439, containing Asn436,in both laccases. Again the presence of a higher degreeof branching in the glycoside moiety of POXA3b isoen-zyme was observed. Analyses led to the assignment of two


Fig. 4. Nucleotide sequence ofP. ostreatus poxa3gene and cDNA deduced amino acid sequence. Introns are shown in lowercase type and indicated byIVS. Putative metal responsive element (MRE), xenobiotic responsive element (XRE), GC, CAAT and TATA boxes are underlined.

N-glycosylation sites in both isoenzymes (Table 3). In fact,Asn256 and Asn436 resulted to be fully modified, whilstno evidence for the glycoside modification of the othertwo putative N-glycosylation sites (Asn89 and Asn220)

was observed. As a fact, Asn220 was found unmodifiedby automated Edman sequencing, whilst the peptide frag-ment containing the last putative glycosilation site at Asn89escaped MALDI-MS analyses.


Table 3MALDI-MS analyses of glycosidic peptides from POXA3a digested withtrypsin and chymotrypsin (A and C) and POXA3b digested with trypsinand chymotrypsin (B and D)

MH+ experimental MH+ theoretical Peptide

A2618.9 2620.5 (428–440)+ Hex5HexNAc2

3230.5 3230.1 (249–265)+ Hex5HexNAc2

3391.1 3391.6 (249–265)+ Hex6HexNAc2

3552.8 3553.4 (249–265)+ Hex7HexNAc2

3714.8 3715.1 (249–265)+ Hex8HexNAc2

B2618.9 2619.5 (428–440)+ Hex5HexNAc2

2781.6 2781.8 (428–440)+ Hex6HexNAc2

3230.5 3230.1 (249–265)+ Hex5HexNAc2

3391.1 3391.6 (249–265)+ Hex6HexNAc2

3552.8 3553.4 (249–265)+ Hex7HexNAc2

3714.8 3715.1 (249–265)+ Hex8HexNAc2

3877.8 3877.2 (249–265)+ Hex9HexNAc2

C3182.8 3183.5 (422–439)+ Hex5HexNAc2

D3182.7 3183.5 (422–439)+ Hex5HexNAc2

3343.4 3344.0 (422–439)+ Hex6HexNAc2

3505.6 3506.1 (422–439)+ Hex7HexNAc2

3.7. POXA3a and POXA3b subunit interactions

As described above, purified POXA3a and POXA3b anal-ysed by SDS–PAGE, gave rise to a pattern constituted bythree components with molecular mass corresponding to 67,18 and 16 kDa (Fig. 1A). POXA3a and POXA3b sampleswere also analysed by SDS–PAGE, but in the absence of�-mercaptoethanol in the electrophoresis sample buffer andwithout any thermal denaturation[26] giving rise to the sameeloctrophoretic pattern already observed. After this treat-ment, however, enzyme activity was only associated, afterdetergent diffusion from the gel[26], to the highest molec-ular mass band.

On native PAGE, both POXA3a and POXA3b showed aunique Coomassie stained band, displaying the same elec-trophoretic mobility (Fig. 1B). On the other hand, when

Fig. 5. Coomassie stained native PAGE of POXA3a and POXA3b samples incubated with urea. (A) POXA3a incubated with 1, 2, 3, 4, 5 M urea (lanes1–5). (B) POXA3b incubated with 1, 2, 3, 4, 5, 6 M urea (lanes 1–6). POXA3a (panel A, lane 6) and POXA3b (panel B, lane 7) were loaded as reference.Arrows indicates high mobility bands.

Fig. 6. Western blot (A) and activity stained native PAGE (B) ofP.ostreatusculture broth samples collected at different growth times.

samples were incubated at increasing concentrations of urea,a different behaviour between the two isoenzymes was ob-served. As a fact, the presence of two bands with higherelectrophoretic mobility was observed in POXA3a samplesincubated in 3, 4, 5 M urea, whilst only the lower mobilityband was always detected for POXA3b even in 6 M urea(Fig. 5).

3.8. POXA3a and POXA3b activation

Western blot analysis and activity stained native PAGE ofculture broth samples, withdrawn at different growth times,was performed loading on both gel electrophoresis 12�l ofeach sample (Fig. 6). In these experiments POXA3a andPOXA3b result to be indistinguishable, since they show thesame electrophoretic behaviour either in native or denatur-ing conditions. The amount of POXA3 proteins reached amaximum after the 3rd–4th day, as shown by the Westernblot analysis, whilst a strong increase in POXA3 activitywas continuously observed at least till the 8th day. Theseresults should indicate a POXA3 specific activity increaseduring the fungal growth.

We have already reported that extracellularP. ostreatusproteases are involved in POXA3 activation[21]. A ser-ine protease inhibitor, PMSF was added to a fungal cultureand POXA3a and POXA3b were purified at the 3rd dayof growth. The specific activities of POXA3a and POXA3b


purified in this condition were about five times and threetimes, respectively, lower than those of the isoenzymes puri-fied in standard condition. On the other hand, the amounts ofpurified isoenzymes were similar to those obtained from cul-tures performed in the absence of protease inhibitor. Thesedata confirm the increase of the specific activities of bothisoenzymes in the extracellular medium during the fungalgrowth, due to a maturation process, which occurs by meansof extracellular proteases. Moreover, it is worth noting thatSDS–PAGE analysis of POXA3a and POXA3b purified fromcultures in the presence of PMSF shows the typical elec-trophoretic pattern consisting of three components.

4. Discussion

It has been reported that a strong increase of laccaseactivity and the production of a new isoenzyme, POXA1b,is obtained inP. ostreatuscopper-supplemented cultures[16,18]. In these cultural conditions two laccase isoen-zymes, POXA3a and POXA3b are produced. SDS–PAGEand MALDI-MS analyses of purified POXA3a and POXA3breveal the presence of three different polypeptides of 67,18 and 16 kDa, whereas the native proteins behave ho-mogeneously as demonstrated by the presence of a singlepeak or band in gel filtration chromatography, isoelectrofo-cusing and native-PAGE analysis. None of the previouslycharacterisedP. ostreatuslaccase isoenzymes show simi-lar behaviour and all of them are monomeric proteins. Onthe other hand, it has been reported that some laccase en-zymes fromPhellinus ribis[27], Trametes villosa[28] andRhizoctonia solani[29] show an homodimeric structure.

The 18 and 16 kDa polypeptides from POXA3a andPOXA3b have a blocked N-terminus. MALDI-MS peptidemapping of the 18 kDa polypeptides from both laccaseisozymes, hydrolysed with either trypsin or chymotrypsin,suggests their identity. Furthermore, the same mass signalswere also found in the peptide maps of 16 kDa polypep-tides both from POXA3a and from POXA3b. A possibleexplanation of these data is that the 16 kDa polypeptidesare generated from the 18 kDa ones, probably through aC-terminal proteolytic processing. The sequence of the fourisolated tryptic peptides from the 18 kDa POXA3a subunit,accounting for about 50% of the entire sequence, did notgive information on the nature of these polypeptides be-cause of the absence of significant homology with otherknown proteins. Furthermore, no sequence encoding thesepeptides have been recognised in the 3′ and 5′ flankingregion of thepoxa3 gene, thus excluding that the smallsubunits could be originated from the maturation of a singlepolypeptide chain containing the largest one.

The protein sequence deduced by cDNA has been veri-fied by means of MALDI-MS mapping against the POXA3aand POXA3b larger subunit. All the putative copper-bindingresidues are present in this sequence, as well as the five Cysresidues found in all the known laccase sequences. Align-

ment of POXC and POXA1b sequences with that deducedfrom the cloned cDNA (poxa3) shows 47 and 46% iden-tity, respectively. It has been reported that two laccases pu-rified from Pleurotus eryngii, separated during the anionicexchange chromatography, are similar in terms of molecularmass, pI, optimum pH and temperature, etc.[29]. Further-more, these proteins have identical N-terminal sequences (12amino acids) showing only two substitutions respect to thecorresponding sequences of POXA3a and POXA3b. There-fore it seems thatP. eryngiilaccases I and II are very similarto P. ostreatusPOXA3a and POXA3b, even if a monomericstructure is suggested forP. eryngiiproteins. Alignment ofPOXA3 sequence with those of other known laccases in databanks reveals the highest identity (67%) withAgaricus bis-porusLac1 and Lac2[30]. TheA. bisporusisoenzymes arevery similar with only 36 differences out of 520 amino acidsin the cDNA deduced sequences. Moreover, SDS–PAGEanalysis of the purified Lac2 shows a predominant band of65 kDa together with lesser amounts of smaller polypep-tides[13,31]. Perry et al.[31], on the basis of Western blotanalyses, suggested that the native Lac2 is a dimer of iden-tical polypeptides, one of which is then partially proteolyti-cally cleaved. Data on the primary structure of the POXA3a18 kDa polypeptide presented here rule out this suggestion,at least inP. ostreatus; no simple explanation for the re-tention of these small polypeptides through the purificationprocess is possible on the basis of the reported results.

Dissociation of the POXA3a complex is observed in 3 Murea whilst POXA3b is not dissociated even in 6 M urea, thusdenoting that some structural differences between POXA3aand POXA3b should affect the intersubunit-binding affinity.However, it has been demonstrated that the binding of thesmall polypeptides to the largest one is not essential forlaccase activity.

A uniquepoxa3gene has been identified but, on the basisof the structural data obtained on the two proteins, it is notpossible to univocally associate this gene to either POXA3aand/or POXA3b. It should be mentioned that the two laccasegenes (lac1 and lac2) identified in A. bisporus, are closetogether with an intergenic region of about 1500 bp[32],whereas the 3′ and 5′ flanking regions (1600 and 1300 bp,respectively) of theP. ostreatus poxa3gene do not showthe presence of other laccase encoding genes. Obviouslythese results do not allow excluding the existence of anotherpoxa3gene close to the already identified gene or locatedin a different chromosomal position.

It has been demonstrated that protease action determinesan increase in POXA3 activity. Further investigations are inprogress to define the role of proteases in modulating laccaseactivity.

Acknowledgments

This work was supported by grants from the Minis-tero dell’Università e della Ricerca Scientifica (Progetti di


Rilevante Interesse Nazionale, PRIN 2002). The authorsthank Prof. Piero Pucci for helpful discussion.

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Atypical laccase isoenzymes from copper supplemented Pleurotus ostreatus cultures

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