Novel Peroxidases of Marasmius Scorodonius

8/3/2019 Novel Peroxidases of Marasmius Scorodonius

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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Novel peroxidases of Marasmius scorodonius

degrade -carotene

Manuela Scheibner & Brbel Hlsdau &

Kateryna Zelena & Manfred Nimtz & Lex de Boer &

Ralf G. Berger & Holger Zorn

Received: 26 September 2007 /Revised: 22 October 2007 /Accepted: 23 October 2007 / Published online: 22 November 2007# Springer-Verlag 2007

Abstract Two extracellular enzymes (MsP1 and MsP2)

capable of efficient-carotene degradation were purifiedfrom culture supernatants of the basidiomycete Marasmius

scorodonius (garlic mushroom). Under native conditions,the enzymes exhibited molecular masses of ~150 and~120 kDa, respectively. SDS-PAGE and mass spectromet-ric data suggested a composition of two identical subunitsfor both enzymes. Biochemical characterisation of the

purified proteins showed isoelectric points of 3.7 and 3.5,and the presence of heme groups in the active enzymes.Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometricab initio sequencing of internal peptides. cDNAs of 1,604

to 1,923 bp, containing open reading frames (ORF) of 508to 513 amino acids, respectively, were cloned from a cDNA

library of M. scorodonius. These data suggest glycosyla-

tion degrees of ~23% for MsP1 and 8% for MsP2.Databank homology searches revealed sequence homolo-gies of MsP1 and MsP2 to unusual peroxidases of thefungi Thanatephorus cucumeris (DyP) and Termitomycesalbuminosus (TAP).

Keywords Basidiomycetes . Carotenoid degradation .

DyP-type peroxidase

Introduction

The degradation of carotenoids has been subject of intenseresearch for several decades. From the perspective ofhuman physiology and nutritional sciences, the centric,symmetric cleavage of carotenoids yielding retinoids hasattracted utmost attention. Retinoids act as vitamins,signalling molecules and visual pigments. A wealth ofdifferent apocarotenoids is derived from the excentriccleavage of the carotenoids polyene chain in various plantand animal species, with the plant hormone abscisic acid

being the best-investigated example (Schwartz et al. 1997).Many of these apocarotenoids (norisoprenoids) act as

potent flavour compounds. Prominent representatives in-

clude - and -ionone, geraniol and -damascenone(Winterhalter and Rouseff 2002).

Apart from the generation of retinoids, plant hormonesor flavour compounds, there is a strong interest of thedetergent and food industries in carotenoid degradation for

bleaching purposes. In bakery, for example, carotenoids aredegraded by the so-called co-oxidation system: Carotenoidsare oxidized by free radical species generated fromlinolenic acid by lipoxygenase catalysis (Wache et al.2003). Mainly soybean extracts containing lipoxygenase

Appl Microbiol Biotechnol (2008) 77:12411250

DOI 10.1007/s00253-007-1261-9

M. Scheibner: K. Zelena : R. G. BergerZentrum Angewandte Chemie,Institut fr Lebensmittelchemie der Universitt Hannover,Wunstorfer Strae 14,30453 Hannover, Germany

B. Hlsdau : H. Zorn (*)AG Technische Biochemie, Universitt Dortmund,Emil-Figge-Str. 68,

44221 Dortmund, Germanye-mail: [email protected]

M. NimtzHelmholtz-Zentrum fr Infektionsforschung,Abteilung Biophysikalische Analytik,Inhoffenstr. 7,38124 Braunschweig, Germany

L. de BoerDepartment of Biochemistry and Nutrition,DSM Food Specialties,P.O. Box 1, 2600 MA Delft, The Netherlands


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isoforms are used to meet consumers demands for a whitecrumb.

Although some 100 million tons of carotenoids arebiosynthesized and subsequently degraded naturally everyyear, surprisingly few data have become known on the bioticcarotenoid degradation by microorganisms (Rodrguez-Bustamante et al. 2007). Mixed cultures of Trichosporon

asahii and Paenibacillus amylolyticus degraded luteinderived from marigold flowers (Tagetes erecta), but theenzymes involved in the degradation pathways have not yet

been characterized (Rodriguez-Bustamante et al. 2005).Marasco et al. (2006) recently identified and cloned gene

homologues of eukaryotic carotenoid cleavage dioxyge-nases in the genomes of different cyanobacteria.

An extra-cellular versatile peroxidase of the ediblemushroom Pleurotus eryngii (previously erroneously clas-sified as Lepista irina) was found to efficiently degrade -carotene (Zorn et al. 2003a). Fungal versatile peroxidasesare key enzymes of natural lignin degradation, which have

been reported to share catalytic properties of lignin peroxidases and manganese peroxidases. In a previousstudy (Zorn et al. 2003b), numerous filamentous fungi andyeasts, which were known for de novo synthesis or bio-transformation of mono-, sesqui-, tri- or tetraterpenes, werescreened for their capability to degrade -carotene. Somestrains discolored a -carotene-containing growth agar,indicating an efficient carotenoid degradation. Using a

photometric bleaching assay, the -carotene cleavingenzyme activities of Marasmius scorodonius were partiallycharacterized. Marasmius scorodonius (CBS 137.83; gar-lic mushroom) is a small edible species, which grows on

wood and further lignified plant materials (Ainsworth et al.1973). Due to its intense garlic-like flavour, it is used as aspice. In the present study, the enzymes catalyzing thecarotenoid degradation were purified to electrophoretichomogeneity, and the encoding genes were cloned fromcDNA and genomic DNA.

Materials and methods

General

The Marasmius scorodonius strain (CBS 137.86) wasobtained from the Dutch Centraalbureau voor Schimmel-cultures, Baarn. Due to the heat and light sensitivity ofcarotenoids, -carotene containing solutions were preparedfreshly before use. The cultivations were performed in theabsence of light, and standard sterile techniques wereapplied. Quantitative data represent average values of atleast duplicate analyses.

Chemicals

The constituents of nutrient media were purchased fromMerck (Darmstadt, Germany). -Carotene and Tween 80(free of peroxides) were obtained from Fluka/Sigma(Taufkirchen, Germany). Solvents were provided by BASF(Ludwigshafen, Germany) and Baker (Deventer, The

Netherlands). All solvents were distilled before use.

Biochemical enzyme characterisation

An enzyme assay (Ben Aziz et al. 1971) was modifiedaccording to Zorn et al. (2003b). Shortly, the time-dependentdecrease of absorbance of an aqueous -carotene emulsionwas monitored at 450 nm using a tempered spectral

photometer. Applicability of the test was checked by dilutingaliquots of the enzyme sample with buffer solution (50 mMsodium acetate buffer, pH 3.5, 27C). A linear correlation

between activity and sample amount was found.

Protein concentration

The protein concentration was estimated by the method ofLowry et al. (1951) using the DC-Protein-Assay (Biorad,Hercules, USA) and bovine serum albumin as a standard.

Electrophoresis

SDS-PAGE was performed by the method of Laemmli(1979) with 4% (w/v) polyacrylamide in the stacking gelsand 16% (w/v) polyacrylamide in the resolving gels.

Proteins were stained with 0.1% (m/v) Coomassie BrillantBlue G/R-250 (Serva, Heidelberg, Germany) or immunos-tained using alkaline phosphatase conjugated anti-rabbitIgG (Invitrogen, Carlsbad, CA). Polyclonal antibodieswere produced in rabbits using standard protocols (Bio-Genes, Berlin, Germany). For immunization, MsP1 andMsP2 were purified, pooled and concentrated from 4 l of

M. scorodonius culture supernatant as described below.An electrophoresis calibration kit (14.5200 kDa; Roth,

Karlsruhe, Germany) was used for the preparation of acalibration curve for determination of molecular masses.

For heme staining with a solution of 3,3,5,5-

tetramethylbenzidine (TMBZ) in methanol according toThomas et al. (1976) and Henne et al. (2001), gels wererun at 4C under non-denaturing conditions.

Isoelectric focussing

The samples were concentrated and desalted using Vivaspin15R ultra-filtration units (MWCO 10 kDa, Vivascience,

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Gttingen, Germany) and subjected to isoelectric focussing polyacrylamide gel electrophoresis (IEF-PAGE) withimmobilised pH gradient. Electrophoresis conditions: geldimensions 12.5 cm12.5 cm0.3 mm; pH range 36(Serva, Heidelberg, Germany); voltage 2,000 V, 6 mA,12 W, 3,500 Vh; sample volume 12 l.

The samples were applied twice, laterally reversed on

both sides of the IEF gel. For detection, the gel was cutconcentric and one half was subjected to Coomassiestaining, the second to activity de-staining. 50 ml of -carotene solution (0.01% m/v+Tween 80 1% m/v), 15-ml

buffer solution (50 mM sodium acetate, pH 6.0), 100 ltrace element solution (containing Fe-, Cu-, Zn- and Mn-ions), and 0.7 g agarose were mixed to give an orangecoloured agarose gel of 2 mm thickness for the de-stainingtest. One half of the IEF gel was covered with this caroteneagar, pinned down and incubated at 27C for 24 h. Toensure the correct assignment of protein bands, thedecoloured spots were marked on the IEF gel by a pinprick

and, after removal of the carotene agar, the gel wasadditionally stained by Coomassie blue.

Enzyme purification by fast protein liquid chromatography

All purification steps were performed in a cooling cham- ber at 6C. A Biologic Duoflow fast protein liquidchromatography system (Biorad) was employed for thechromatographic protein separation. The protein concen-tration of the eluate was monitored at =280 nm, andenzyme activity was determined in all protein containingfractions.

M. scorodonius was maintained on a -carotene-containinggrowth agar as described previously (Zorn et al. 2003b).For preparation of precultures, 14-mm diameter agar plugsfrom the leading mycelial edge were transferred into100 ml of standard nutrition solution (30 g l1 glucose1H2O; 4.5 g l

1 asparagine 1 H2O; 1.5 g l1 KH2PO4; 0.5 g

l1 MgSO4; 3.0 g l1 yeast extract; 15 g l1 agar-agar; 1 ml

l1 trace element solution containing Cu, Fe, Zn, Mn andEDTA; pH adjusted to 6.0) and homogenized using anUltra Turrax (IKA, Staufen, Germany). After cultivationfor 7 days at 24C and 150 rpm, the cultures werehomogenized and 20 ml of the pre-cultures were trans-

ferred into 500-ml Erlenmeyer flasks containing 250 ml offresh standard nutrition solution.

Supernatant was collected from the submerged culturesafter 4 days, separated from the mycelium by filtration orcentrifugation (4,000g, 1 h, 4C), and concentrated bytangential flow filtration (Vivaflow 200, 10 kDa MWCO;Vivascience). The retentate was subjected to ion exchangechromatography (IEC).

Ion exchange chromatography

A Q-Sepharose high-performance column (25 ml, Pharma-cia Biotech, Uppsala, Sweden) was used for ion exchangechromatography. Sodium acetate (50 mM, pH 6.0) servedas start buffer, and the enzymes were eluted with a lineargradient of 0100% sodium acetate buffer containing 1 M

sodium chloride. The flow rate was 3 ml min1, fractionswere collected every minute. The active fractions were

pooled and concentrated by ultra filtration (see above).

Size exclusion chromatography

A Superdex 200 HR 10/30 column (Pharmacia Biotech)with a bed volume of 24 ml and a separation range of 10 to600 kDa was used. The elution buffer consisted of sodiumacetate (50 mM, pH 6.0; flow rate 0.5 ml min1). Fractionswere collected every 2 min. A calibration curve was

prepared with reference proteins (HMW and LMW Gel

Filtration Calibration Kit, Pharmcia Biotech) for calculatingthe molecular mass.

ElectrosprayIonization tandem mass spectrometry

ESIMS/MS analyses were performed as reported previ-ously (Zorn et al. 2005). Protein spots were excised fromCoomassie stained SDS-PAGE gels and digested withtrypsin. The resulting peptides were extracted and purifiedaccording to standard protocols. A QTof II mass spec-trometer (Micromass, Manchester, England) equippedwith a nanospray ion source and gold coated capillaries

(Protana, Odense, Denmark) was used for electrospray MSof peptides. For collision induced dissociation experi-ments, multiple charged parent ions were selectivelytransmitted from the quadrupole mass analyzer into thecollision cell (collision energy 2530 eV for optimalfragmentation). The resulting daughter ions were separat-ed by an orthogonal time-of-flight mass analyser. Theacquired MSMS spectra were enhanced (Max. Ent. 3,Micromass) and used for the de novo sequencing of tryptic

peptides. The fasts3 algorithm, which compares linked peptides to a protein databank, was used for homologyqueries.

MALDITOFMS

Mass spectra were recorded on a Bruker ultraflex TOF/TOF mass spectrometer (Bruker Daltonics, Bremen,Germany) at an acceleration voltage of 20 kV in a linearmode. In a dried-droplet application, sinapic acid servedas matrix.

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UV/Vis-spectroscopy

Absorption spectra were recorded using a Perkin ElmerLambda 12 (berlingen, Germany) spectral photometerequipped with a tempered cell holder and magneticstirrer.

cDNA synthesis and PCR-screening

For cloning of the MsP1 and MsP2 encoding cDNAsequences, a cDNA library of M. scorodonius wasconstructed and screened by polymerase chain reaction(PCR). Mycelium of M. scorodonius was harvested at anenzyme activity of ~0.5 mU ml1 on the fourth cultureday. Cell disruption was achieved by grinding themycelium (210 mg) under liquid nitrogen. For isolationof total RNA, a silica-gel-based membrane (RNeasy PlantMini Kit, Qiagen, Hilden, Germany) was applied. Integrityof the RNA was checked by denaturing formaldehyde

agarose gel electrophoresis and ethidium bromide stain-ing. cDNA was synthesised with the SMART cDNAlibrary construction kit (BD Biosciences, Heidelberg,Germany) according to the manufactures instructions.Super Script II RNase H reverse transcriptase (GIBCOBRL, Life Technologies, Paisley, Scotland) was used forfirst-strand synthesis. Primer construction was performedwith the assistance of the primer3 algorithm (Rozen andSkaletsky 2000), and PCR primers were synthesized byRoth (Karlsruhe, Germany). For PCR reactions, ~20 ng ofgenomic DNA or cDNA, respectively, were used astemplate in 20-l reaction mixtures containing 1 PCR

Buffer (QIAGEN), 0.2-mM dNTPs, 3.0-mM MgCl2, 0.5-Mof each primer, and 0.5-U HotStarTaq DNA polymerase(QIAGEN). Amplification experiments were performedin a Master Cycler gradient (Eppendorf, Hamburg,Germany). The following primers were used for ampli-fication of full-length cDNAs of MsP1 and MsP2: MsP1forward (5>ATG AAG CTT TTT TCT GCC TCCCTA GAC TGA AAG CAC AGTCCT GAT CGAGT ATG CGGCTC ACT TAC CTT CCTCAAAC AGA AAG CGT GTT CTG GAT CGACT GCG AGTGAA GAG GGA AATTG CACGTC AGA ATC GCT AC


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MsP1 and MsP2 by primer walking. Based on the obtainedsequence information, three different full-length cDNAswere amplified for MsP1, and two for MsP2.

The cDNAs of MsP1 (1,6041,682 bp) contained openreading frames (ORF) of 508 or 513 amino acids,respectively, while the mature proteins consisted of 453and 458 amino acids. A molecular mass of ~49 kDa wascalculated for the mature proteins. All of the tryptic

peptides of MsP1 sequenced by ESI-tandem mass spec-trometry were present in the translated protein sequences.The amino acid sequences contained eight potential N-glycosylation sites and one potential O-glycosylation site.

The cDNAs of MsP2 (1,857

1,923 bp) comprised anopen reading frame (ORF) of 1,533 bp, corresponding to a510 amino acid protein. 3 non-coding regions of 279345 bp were identified in the MsP2 cDNAs. For the mature

proteins (453 AA), a molecular mass of ~48 kDa wascalculated. Four potential N-glycosylation and three poten-tial O-glycosylation sites were detected in the amino acidsequences. The translated amino acid sequences containedall of the peptides derived from sequencing of MsP2 byESI-tandem mass spectrometry. The overall amino acidsequence homologies of MsP1 and MsP2 to the enzymesDyP (accession number Q8WZK8) and TAP (Q8NKF3)

produced by Thanatephorus cucumeris and Termitomycesalbuminosus, respectively, were 48%, and the sequenceidentity between the MsP1 and MsP2 enzymes was 63%.

Cloning of MsP1 and MsP2 from genomic DNA

Amplificates of 2,093 and 2,135 bp were obtained forMsP1 and MsP2, respectively, when genomic DNA of M.

Fig. 2 SDS-PAGE analysis ofactive fractions (16 to 19) iso-lated by size exclusion chroma-tography; Ref., reference

proteins; left, silver staining;right, Western blot

Fig. 3 a: UV-Vis absorptionspectrum of purified MsP2; b:SDS-PAGE analysis of MsP2with heme staining using TMBZ



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scorodonius was used as a template in the PCR reactions.Both genes contained ten introns of 50- to 83-bp length.

Discussion

Based on the overall sequence homologies of ~50% on theamino acid level, the novel peroxidases MsP1 and MsP2may be assigned to the group of DyP-type peroxidases(dye decolorizing peroxidases). DyP represents a secreted,iron-containing enzyme of the class II plant peroxidases(classification according to Welinder 1992), which wasisolated from the fungus Thanatephorus cucumeris Dec1(Sugano et al. 1999). As DyP did not reveal sequencehomologies to other class II enzymes, it was regarded as the

prototype of a new peroxidase family (Sugano et al. 2000).This family comprises currently about 170 proteins from

eukaryotes and prokaryotes. Among the 13 eukaryoticmembers, nine are fungal proteins (InterPro at EMBL-EBIweb site; Faraco et al. 2007), including a phenol oxidasefrom Termitomyces albuminosus (TAP) and a peroxidase ofa Polyporaceae sp. (PoP).

The ability to oxidize phenolic compounds, such as 2,6-dimethoxyphenol and guaiacol, in the absence of Mn2+

distinguishes DyP from manganese peroxidases. DyP doesnot degrade non-phenolic compounds, thus differing fromlignin peroxidases. Another interesting characteristic ofDyP is its ability to degrade synthetic anthraquinone dyes,which are not oxidized by most other peroxidases (Kim and

Shoda 1999).Different from lignin peroxidases, MsP1 and MsP2 do

neither contain the conserved tryptophan residue, which isessential for the oxidation of veratryl alcohol and furtheraromatic substrates (Blodig et al. 1998; Choinowski et al.1999), nor the two Ca2+binding sites that typically stabilizesecreted peroxidases (Conesa et al. 2000; Martnez 2002).Likewise missing is the manganese binding site ofmanganese and versatile peroxidases (Kishi et al. 1996,Whitwam et al. 1997, Sundaramoorthy et al. 1997).

MsP1 and MsP2, like most other members of the DyP-type family, exhibit one conserved asparagine and fourconserved histidine residues, which are presumed to beessential for the functionality of the enzymes (Fig. 4). ForDyP, His164 and Asp278 have been shown to be involvedin the binding of the heme group by X-ray diffraction (Satoet al. 2004), and additionally by site-directed mutagenesis

(Sugano et al. 2004). A recombinant and deglycosylatedenzyme expressed in A. oryzae was employed in theseinvestigations.

By MALDITOF analyses, molecular masses of 64.2 kDaand 52.4 kDa, respectively, were determined for MsP1 andMsP2. These data suggest glycosylation degrees of ~23% forMsP1 and 8% for MsP2. Similar carbohydrate contents have

been reported for DyP (17%; Sugano et al. 1999) and TAP(1528%; Johjima et al. 2003). Carbohydrate side chainsare typically not required for the catalytic activity of fungalextracellular peroxidases (Sato et al. 2004; Nie et al. 1999),

but they have been shown to protect the enzymes from

degradation by peptidases and to increase the enzymesthermostability (Lis and Sharon 1993, Rehm 2002; Suganoet al. 2000).

While characteristic signal peptide cleavage sites wereidentified in position 20/21 (MsP1) and 19/20 (MsP2;SignalP 3.0, Bendtsen et al. 2004), no typical cleavage siteswere identified before the N-termini of mature MsP1 andMsP2, DyP and TAP. Presumably, the secreted proteins

post-translationally undergo a two-step processing. As DyP,heterologously expressed in Aspergillus oryzae, showed thesame N-terminus as the wild-type enzyme, catalysis of thesecond hydrolysis step by the peroxidase itself was

discussed (Sugano et al. 2000). However, an experimentalproof of this hypothesis is still missing.

An uncommon characteristic of MsP1 and MsP2 is theirnative occurrence as dimers. An extracellular dimeric perox-idase has so far solely been described for the basidiomycete

Pleurotus ostreatus (Kang et al. 1993). The isolelectricpoints of MsP1 (3.7) and MsP2 (3.5) are close to the onereported for DyP (3.8; Kim and Shoda 1999). AlthoughSDS-PAGE analysis indicated electrophoretic homogenous

proteins in the active fractions of the final chromatographic purification step (cf. Fig. 2), IEF electrophoresis withCoomassie and activity de-staining revealed additional faint

protein bands. This observation may either be explained byvarying glycosylation patterns (Lis and Sharon 1993), or bythe presence of iso-enzymes. The multiple cDNAs encodingMsP1 and MsP2 with sequence identities of up to 98% wellexplain the protein pattern observed on the IEF gels. The

production of several iso-enzymes is a typical characteristicof basidiomycetous fungi. Altogether, ten genes encodinglignin peroxidases and five genes encoding manganese

peroxidases have been identified in the genome of thewhite-rot fungus Phanerochaete chrysosporium (Hoegger et

Table 2 Amino acid sequences of tryptic peptides determined byESItandemMS (L symbolises L orI)

Enzyme Peptide sequences

MsP1 GGEYFFSPSLSALR QLPYGPEVTDAEK

NSGTDAPNTPRMsP2 DGSFLAFR

PQNTNNHLLRLVLNTDFVSSRFLADNAALTQGNADLLGARSGAPVDLAPTADDVDLANDPQR



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TAP MQLK--NFLAATAAFVALSQPIAAYHV--KRARSSPLIVPFPGQGALPTAAQVQSTSAGD 56

PoP MQLK--HFLAATAAFSAVTQSAFAYHV--KRARSSPLIGSFPGQPPLPTIAEVQSSSAGN 56

DyP MRLS--LFVVSVAVLVGSSSHVNAAKLGARQTRTTPLLTNFPGQAPLPTLTQHTTESGAN 58

MsP1 MKLFSASVFAAIVASHYASATAHIRAPNVKPRRTNSLLITPPQQPPLPSAQQAASASS-- 58

MsP2 MRLTYLPLFAGIAIQSACAFPNFSKSSILKPRRTNSLLINPDAQPDLPTAKQASTAAA-- 58

* * * * * **

TAP DS-IPFENIQADILVGMKKQNEKFVFFHINNAATFKSVLKTYAPANITSVATLIGPISAQ 115

PoP DS-LPFENIQGDILVGMKKDKEKFVFFHINNATTFKSFLKTYAPANITSVQTIIGPASGQ 115

DyP DTILPLNNIQGDILVGMKKQKERFVFFQVNDATSFKTALKTYVPERITSAAILISDPSQQ 118

MsP1 SAGLNLTDIQGDILIGMKKNKELFFFFSVTDAATFKAKLGSDILGLITSTDQLLANDT-Q 117

MsP2 SVGLNLTDIQGDILIGMKKNKEMFFFFSIADAAAFKSHLGSAILPLITSTQQLLAVAS-Q 117** *** **** * * ** * ** * *** *

TAP PQAVVNVAFSQAGLGALGVTDN-LGDTAFTGGQFADASDGLGDDTN-TWEPAFKGTNIDG 173

PoP PQAFVNLAFSHTGFGALGVADD-LQDTAFTAGQFADAPS-FGDDTS-TWEEAFKGTNVDG 172

DyP PLAFVNLGFSNTGLQALGITDD-LGDAQFPDGQFADAAN-LGDDLS-QWVAPFTGTTIHG 175

MsP1 PVTAVNVAFSSTGLKALGITDD-LKDPVFEAGMLSNAVSDLSDPGTGNWVPGFVGTSVHG 176

MsP2 PTTAVNLAFSQTGLNALGLAAQGLGDSLFASGQFSGAQS-LGDPGTSNWVQAFAGTGIHG 176

* ** ** * *** * * * * * * * * ** *

TAP VFLIISDQDSIITQYQDDLQAKLGDAWTVVYDLSGAARPGDQFGHEHFGYLDGISNPLIK 233

PoP VFLIGSDDVTTTNQYRDDLKAKLGDAWTVLLDLDSAARPGAEKGHEHFGYLDGISNPTIP 232

DyP VFLIGSDQDDFLDQFTDDISSTFGSSITQVQALSGSARPGDQAGHEHFGFLDGISQPSVT 235

MsP1 VFLLASDTIDNVNTELANIQTILNGSITEIHRLQGAARPGDQQGHEHFGFMDGISNPAVD 236

MsP2 VFLLASDTVDNVNAELSQIQSILGTSITEAYRLQGEARPGDQQGHEHFGFMDGISNPAID 236

*** ** * * **** ****** **** *

TAP GFG---EPLPGQAFIDPGIILVGRANDTVTT-RPAWALDGSFLAFRKLKQLVPEFHKYTL 289

PoP GFG---TPFPGQAVVDSGVIFAGRTNDPVTN-RPSWALDGSFLVFRKLKQLVPEFHKWTL 288DyP GWET--TVFPGQAVVPPGIILTGRDGDTGT--RPSWALDGSFMAFRHFQQKVPEFNAYTL 291

MsP1 GFTPPAEIRPGQALIPPGIMLLGEANDTFQNDRPPWAKDGSFLVFRQMQQRAPEFNKFLQ 296

MsP2 GFS---TALPGQAVLSPGLFLLGEDGDGSSSSRPSWAKDGSFLAFRQLQQRVPEFNKFLA 293

* **** * * * ** ** **** ** * ***

TAP DNALQ-NQSGNLSTEEGALLLGSRMFGRWNSGAPIDLTPDVDDPALGNDPNRNNNFNYIH 348

PoP DNALQ-NQAGNLTVEEGALLLGSRMFGRWNSGAPIDLTPDVDDPTLGNDPQRNNDFNYIH 347

DyP ANAIPANSAGNLTQQEGAEFLGARMFGRWKSGAPIDLAPTADDPALGADPQRNNNFDYS- 350

MsP1 DHALN---MPNMTSEQGADLLGARIVGRWKSGAPIDLTPLVDDPVLAADNQRNNNFDFSD 353

MsP2 DNAA--------LTQGNADLLGARMMGRWKSGAPVDLAPTADDVDLANDPQRNNNFNFTH 345

* * ** * *** **** ** * ** * * *** *

TAP PGEDPATDQSRCPFTAHIRKTNPRDLESQNLIPEFFHAIRAGTPYGPEVSYAESSSNTTQ 408

PoP PGEDLTTDETRCPFTAHVRKTNPRDLEAQGLIPDLFHAIRAGTPYGPEVTDAESNSNTTS 407

DyP ---DTLTDETRCPFGAHVRKTNPR--QDLGGPVDTFHAMRSSIPYGPETSDAELASGVTA 405

MsP1 A-----TNQTRCPFSAHIRKANPRGDLGGINKFPNQHIIRAGIPYGPEVTDAEKASNSSS 408

MsP2 AGFTETTDETHCPFSAHIRKTNPRSDFNPQN--TNNHIIRAGIPYGPEVTDAEASSNTSS 403

* *** ** ** *** * * ***** ** *

TAP ID----RGLAFVEYQSNISNGFRFQQVNWANNNKFPFNKSISEPGLDPVIGQDK------ 458

PoP ID----RGLAFVEYQSVISNGFRFQQLNWANNANFPFNKSEP-LGLDPVIGQG------- 455

DyP QD----RGLLFVEYQSIIGNGFRFQQINWANNANFPFSKPIT-PGIEPIIGQT------- 453

MsP1 TDPSLERGLAFVAYQSNIQNGFVFLQKNWVDNTNFFR----PGTGVDPLIGTNSRNSGTD 464

MsP2 TDASLERGLAFVAYQSNIGNGFAFIQQNWVDNANFFFG-KTTPPGIDPIIGSNAAQN-NF 461

* *** ** *** * *** * * ** * * * * **

TAP --GANRITTGLNPLNVSESLSIP-DFIVSNGGEYFFAPSITSIVETLAA 504

PoP ----TRQTFGLDPRNASDSLTIP-QIIISNGGEYFFSPSITALVEHFGA 499

DyP ---TPRTVGGLDPLNQNETFTVP-LFVIPKGGEYFFLPSISALTATIAA 498

MsP1 APNTPRVVSGLDPNNATSTIEIDIDFVVSRGGEYFFSPSLSAIRTVLSV 513

MsP2 APNSPRPVSGLDPTDSTTIVTLNTDFVVSRGGEYFFSPSLSAIQNTLSV 510

* ** * ****** **

Fig. 4 Alignment of the enzymes TAP (T. albuminosus), PoP( Polyporaceae sp.), DyP (T. cucumeris), MsP1 and MsP2 (M.

scorodonius); (asterisk, conserved amino acid residue; filled square,histidine presumably involved in the binding of the heme group; filled

triangle, asparagine residue presumably involved in the binding of theheme group; arrow up: conserved histidine residues); N-terminal

amino acids shaded; alignment was performed with CLUSTAL W(Higgins et al. 1994)

Fig. 5 Comparison of the organisation of the MsP1 and MsP2 genes of M. scorodonius; black area, Introns, white area, Exons; two uncommonshort exons (CCCC, GCCT) marked with arrows



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al. 2007; Martnez 2002). The basidiomycete Pleurotuseryngii secretes three versatile peroxidase iso-enzymes withhigh sequence homology (Ruz Dueas et al. 1999), and upto 16 lignin peroxidases with pIs of 3.1 to 3.7 were formed

by Trametes versicolor(Johansson and Nyman 1996).The MsP1 and MsP2 encoding genes both contain ten

introns. Similarly, a gene encoding a putative DyP-type

enzyme with nine introns has been cloned only recentlyfrom a Pleurotus ostreatus genomic library (Faraco et al.2007). The intronexon structure of MsP1 and MsP2 israther similar (Fig. 5).

All introns of MsP1 and MsP2 are flanked by a GTdinucleotide at the 5, and by AG at the 3 ends. While thisorganization is typical for 9899.9% of the introns of thefungi Saccharomyces cerevisiae, Schizosaccharomyces

pombe, Aspergillus nidulans, Neurospora crassa andCryptococcus neoformans (Kupfer et al. 2004), twouncommon short exons of only four nucleotides are presentin the MsP2 gene.

Acknowledgement Support of the work by the Deutsche For-schungsgemeinschaft (ZO 122/12) is gratefully acknowledged. Theauthors thank O. Scheibner (HKI Jena, Germany) for the MALDITOF analyses.

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Novel Peroxidases of Marasmius Scorodonius

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