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A novel type of peroxidase gene from the white-rot fungus Trametes versicolor
Btl
Biochi~ic~a et B i o p h y s i c a A~ta
ELSEVIER
Biochimica et Biophysica Acta 1207 (1994) 255-259
Short Sequence-Paper
A novel type of peroxidase gene from the white-rot fungus Trametes versicolor 1 Leif J6nsson, Helen G. Becker
2, P e r O l o f N y m a n
*
Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S-221 O0Lund, Sweden
Received 27 January 1994
Abstract
The wood-decaying fungus Trametes versicolor secretes a large number of peroxidase isozymes, presumed to partake in the degradation of lignin. From enzymic studies, two types of peroxidases have been distinguished: lignin peroxidases and manganese peroxidases. We here report the finding of a T. versicolor peroxidase gene, PG V, which displays several features not observed in previously studied peroxidase genes from white-rot fungi, such as a high number of introns (12). Eight of the 12 introns have positions equivalent to introns of peroxidase genes from another white-rot fungus, Phanerochaete chrysosporium. The gene structure of PG V appears to be primarily related to known lignin peroxidase genes, while the encoded mature 339-residue protein has several characteristics in common with manganese peroxidases. Analyses further indicate that PG V encodes a Ser instead of an Asn at a position regarded as invariant within the enzyme superfamily, with the side chain involved in hydrogen bonding with the distal His. Key words: Nucleotide sequence; Peroxidase gene; Lignin biodegradation; T. versicolor
The basidiomycete Trametes (Coriolus) versicolor has often been studied as an efficient degrader of wood [1,2]. T. versicolor secretes large amounts of a copper-containing phenoloxidase, laccase, which has been ascribed an important role in the ligninolytic action of the fungus. In addition, a large number of peroxidase isozymes have been shown to be secreted. In this regard, T. versicolor resembles another, extensively studied, white-rot fungus, Phanerochaete chrysosporium [3], which, however, does not produce any laccase [4]. The extracellular heme-containing peroxidases have been divided into two families, displaying differences in substrate specificity. Lignin peroxidases have been found to oxidize aromatic substrates, while manganese peroxidases have been suggested to act by oxidizing Mn(II) to Mn(III), which then in turn oxidizes phenolic lignin substructures. For both T. versicolor and P. chrysosporium, multiple genes have been shown to be a major source of the large
1The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number X77154. 2 Present address: Department of Chemical Enzymology, Chemical Faculty, M.V. Lomonosov Moscow University, Russia 117234. * Corresponding author. Fax: + 46 46 104534. 0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4838(94)00083-S
number of peroxidase isozymes observed. From P. chrysosporium, genes encoding lignin peroxidases and manganese peroxidases have been characterized (reviewed in [4]). From T. versicolor, several genes proposed to encode lignin peroxidases have been reported [5-7]. Of these sequences, the gene LPGI was found to encode a protein identical with an abundantly expressed lignin peroxidase isozyme, sequenced by Edman degradation of tryptic peptides to an extent of approximately 40% [8]. Analyses of amino-terminal ends of lignin peroxidases and manganese peroxidases isolated from T. versicolor indicate that a high degree of similarity between these enzymes can be expected [8]. Here, we report the finding of a T. versicolor peroxidase gene which displays unique features. The segment of DNA, the sequence of which is shown in Fig. 1, was obtained from a genomic library of T. versicolor, as described in [5]. The gene, P G V, is predicted to contain 12 introns. All previous peroxidase genes from T. versicolor contain six introns, identically positioned [7]. These six introns are also present in PG V as the three first and the three last introns (Fig. 2A). Of the remaining six introns in P G V, four have positions identical with LIP2 in Fig. 2A, which is the gene for a major lignin peroxidase isozyme (H2) in P. chrysosporium [3,9].
256
L. Ji~nsson et aL / Biochimica et Biophysica Acta 1207 (1994) 255-259
CTCGAGGTTGCCAAAA•GT•AGGA•ATC•AGTTCT•TGCCGGT•TCCCACTCATCGCA•AATGGCCTT•AAGACACTTGCGAGCTTCGTCTCTGT•CTTG MetAlaPheLysThrLeuAlaSerPheValSerValLeuA
i00
CCGCCCTCCAGGTCGC~AGCGGTCTGTTCTTCT~CTTCTCTACTCGTTTTCAACCTGCTGACATTGCACCAGGTGCC~TTAC~CGC~TCGCTTGCCC 200 laAlaLeuGlnValAlaSerG
lyAlaLeuThrArgArgValAlaCysPr
CGACGGCGTGAA•ACGGCTAC•AACGCGGCGTGCTGC•AGCTCTTCGC•GTCCGCGATGATAT•CAGAAGAAC•TGTTCGA•AACGGCGAGTGCGGTGAG
300
•AspG•yVa•AsnThrA•aThrAsnA•aA•aCys•ysG•nLeuPheA•aVa•ArgAspAspI•eG•nLysAsnLeuPheAspAsnG•yG•u•ysG•yG•u GACGTCCACGAGTCC~T~CGT~T~ACCTTCCACGA~GCCATTGGCTTCT~CCGCTCTGCGGAGC~TAA~GGCACCTTCGGGTAGGTCCTTGCACCTATCA AspVa•HisG•uSerLeuArgLeuThrPheHi•AspA•aI•eG•yPheSerArgSerA•aG•uA•aAsnG•yThrPheG•
400
CGACGT~GAAGCGTTGCTGAATC~TT~TCTTTACAC-CGGCGGAGGCGCGGA~C-C-CT~GATCTCCAT~TT~GCGTCCATCGAGACTAA~TTCCACGCGAGT yGlyGlyGlyAlaAspGlySerIleSerIlePheAlaSerIleGluThrAsnPheHisAlaSer
500
CTCGGTATCGA•GAGATCGTCGGCGAGCAGGCACCCTTCATTGC•CGC•A•AA•CT•A•CGTTGGCGACTTGTACGTCTTCTCCTGCCATACTTGGAAGT LeuGlyIleAspGluIleValGlyGluGlnAlaProPheIleAlaArgHisAsnLeuThrValGlyAspPh
600
TGTT•TAACTCGGAATCGTGCAGCATC•AGTTTGCTGGAGCCGT•GGTGTCAGCAACTGC•CCGGCGCCCCGCGCCTGCAGTTCCTGCTTGGTGAGTGGG eIleGlnPheAlaGlyAlaValGlyValSerAsnCysProGlyAlaProArgLeuGlnPheLeuLeuG
700
CAGGTCTCCGAATGTCTGGCTCTTTA•TGAACGC4•ATCTATCT•AGGC•GTCCCAACGCCA•T•AAC••GCGcCCGA•AAGACCATCCCGGAGCCATTCG lyArgProAsnAlaThrGlnProAlaProAspLysThrIleProGluProPheA
800
GTAAGAGATAAGAAACATTCCC-C~C~GTAAGGCGGTACTGACGCACTTTCAACACTAGACACCGT~GACTCCATTCTCGCCCGTTTCCTGGACGCCGCTG900 spThrValAspSerIleLeuAlaArgPheLeuAspAlaAlaA
ACTT~AC.C~CGC~GGAGGTCGTCGCTCTGCTGGCGTCGTATGTACTCTTCACGCTCGTCGGTTCACTCGATTACTGACCAGCACGTTTGTAGGCACACCA spPheSerProAlaGluValValAlaLeuLeuAlaSe rHisThrI
i000
T~C.~CTC-CGC-CCGATGAGGT~GA~GA~GATTC~CGG`a~k~TT~GAcT~CACGCCCGAGCTTTTCGACA~c~AGTTCTTCATCGAGA~T~AGCTGCGii00 •eA•aA•aA•aAspG•uVa•AspPr•ThrI•ePr•G•yThrPr•PheAspSerThrPr•G•uLeuPheAspThrG•nPhePheI•eG•uThrG•nLeuAr
CGGCACTGGGTT•CCTGGGTACGTTGTCGTTTGCTATGGCGCGAAACGTTACTGATGTAGAGCAGAACGGCCGGCAACCAGGGCGAGGTTCTGTCCCCTC qGlyThrGlyPheProGl yThrAlaGlyAsnGlnGlyGluValLeuSerProL
1200
TTCCCGGAGAGATGCGC•TGCAGTC•GACTCCGAGGTAAGATCAAGCTTCGGCCGTTGTGTTCTTGCACTCAACAAATCTTTTTCTAGCTCGCTCGTGAC euProGlyGluMetArgLeuGlnSerAspSerGlu LeuAlaArgAsp
1300
T~GGA~TGC~TC-CGAGTGC~AGTCCATGGTCAGTAAGTATTGCTCATTTGCTCTAT`a~kTCGACTATGTCCTGATTCATGTTTCCTTAGACAACCAGTC 1400 SerArgThrAlaCysGluTrpGlnSerMetValA
snAsnGlnSe
GAAGATGATGA~GGC~TTCGCGC~TGCCATGGCTAAGCTCGCGGTCATCGGCCAGGA~GTCAGCCAGTTGATTGACTGCTCCGAAGTGGTGCGTCCTCAC rLysMetMetThrA•aPheA•aA•aA•aMetA•aLysLeuA•aVa•I•eG•yG•nAspVa•SerG•nLeuI•eAspCysSerG•u•a•
1500
ACATGCCTTGCGTCT~TGACACGTTGAC~AATTTCTTCGCGAGTAGATCCCGATGCCC.C~GCC~CC-~GAGCGCCGCGCACTTCCCCGCCGGACTTAGC IleProMetProProProProAlaSerAlaAlaHisPheProAlaGlyLeuSer
1600
AACGCGGACGTCGAGCAGGCTGTGCGTGTTTCGATGTTGATGGTCTTCCCCATGTTGCTCACGTATGCCGATATAGTGCGCCGAGACCCCCTTCCCGACT AsnAlaAspValGluGlnAla CysAlaGluThrProPheProThr
1700
CTC•AGA•CGAC•••GGA••CGAGACCTCC-GTGC.CCCCTGTGTAAGTGA•T•CAC-GGCTATCTCTCACCTGCTAGGCTCTAACTCTTGGATCTCTCTCTA LeuGlnThrAspProGlyProGluThrSerValAlaProVa
1800
GCCCCCC GTCTTAAC-CTAAGCACAGGAGATGTCGGCGGAAC-CTCTGC TATAGCGAGT GC TT TGCT TATTTAGAAATGGTCGTT TGT TACTAT IProProSerEND
1892
Fig. 1. The nucleotide sequence of PG V. The DNA segment shown is from a 3 kbp XhoI-fragment which was inserted into pGEM-TZf( + ). The segment shown was sequenced in one direction using a series of nested deletions [17], and in the other direction by the primer synthesis approach. Standard methods were employed for cloning and dideoxy-sequencing [18]. Computer analyses of sequences were performed using the GCG programs (Version 7, 1991, Genetics Computer Group, Inc., Madison, Wisconsin, USA). The predicted translation product is indicated in the figure. The sequence has been submitted to the EMBL/GenBank Database (accession number X77154).
L. Jfnsson et al. / Biochimica et Biophysica Acta 1207 (1994) 255-259
As previously shown [5], four of the six introns in LPGI are positioned identically with introns of P. chrysosporium peroxidase genes. The corresponding figures for PG V are eight introns in common out of twelve (see Fig. 2A). Fig. 2B shows that the intron positions in PG V differ considerably from those reported for a manganese peroxidase gene [10]. Thus, on the gene level, PG V appears related to lignin peroxidases. The translation initiation environment of PG V (CACAATGGC) is in full agreement with the consensus for filamentous fungi [11] (CAMMATGNC, where M = A or C), indicating that PG V may be expressed. The gene is predicted to encode a 339-residue mature protein, preceded by 26 residues. These residues are proposed to be removed in two proteolytic steps, the first accomplished by a signal peptidase and the second by a Kex2-related endopeptidase. Such an arrangement conforms with previously studied lignin peroxidase genes [4,7], while the manganese peroxidase genes from P. chrysosporium, so far studied, do not encode the two basic residues of the second proteolytic cleavage site. Previously known T. versicolor peroxidase sequences are clearly more similar to lignin peroxidases from P. chrysosporium (over 60% identity between encoded mature proteins) than to manganese peroxidases (47-50% identity). PG V, however, does not follow this pattern. The identity with P. chrysosporium lignin peroxidases is roughly the same as with manganese peroxidases. P. chrysosporium manganese peroxidases display a stronger similarity to the translation product of PG V (56-57%) than to lignin peroxidases (50% or less, even within P. chrysosporium). In Fig. 3, it can be seen that for several positions conserved in lignin peroxidases, PG V instead conforms with manganese peroxidases. The most significant difference in amino acid composi-
A. LPGI PGV
LI Pfl LG2
B° /:~V
I
I
I I
~ I
.~NP1 I I I I Fig. 2. Comparison of intron positions. In (A) PG V is compared with LPGI (lignin peroxidase gene from T. versicolor), LIP2 [9] and LG2 [19] (lignin peroxidase genes from P. chrysosporium). In (B) PG V is compared with MNP1 [10] (a manganese peroxidase gene from P. chrysosporium ).
257
tion between T. versicolor lignin and manganese peroxidases has been reported to be the content of Ser [8]. The high content of Ser (24 residues) in the mature protein coded for by PG V is consistent with the reported value for manganese peroxidases (approx. 24 Ser, compared with 16 for lignin peroxidases). The amino-terminal sequence encoded by PG V is also more similar to manganese peroxidases isolated from T. versicolor than to lignin peroxidases. Of the 35 terminal residues reported for manganese peroxidase isozyme TvMP2 [8], only 2 are mismatches when compared with the sequence encoded by PG V. Preliminary studies of a presumed gene for isozyme TvMP2, isolated in our laboratory (Johansson, T., personal communication), indicate that a high degree of identity is not restricted to the amino-terminal portion. Residues interacting with reducing substrates have not been conclusively identified for either lignin or manganese peroxidases. Two Phe in horseradish peroxidase, a structurally related peroxidase [12], have been shown to interact with aromatic substrates [13]. The corresponding residues in a translation product of PG V would be 149 and 150. In PG V, one of these residues, a Phe, is identical with lignin peroxidases, while the other one, an Asp, is identical with manganese peroxidases (Fig. 3). Based on model building studies, residues interacting with an aromatic substrate have been suggested for lignin peroxidase [14] (corresponding to residues 82 (His), 85 (Ile), 148 (Phe), 184 (Val), and 222 (Gln) from PcLG2 in Fig. 3). Residues in manganese peroxidase that may interact with Mn(II) have also been suggested [15,16] (corresponding to residues 35 (Glu), 39 (Glu), and 179 (Asp) [15] or 84 (Asp), 85 (Asp), 179 (Asp), and 182 (Asp) [16] of the manganese peroxidase sequences in Fig. 3). The residues encoded by PG V do not show any conspicuous deviations from the substrate binding sites proposed for either lignin peroxidase or manganese peroxidase, and are fully consistent with the Mn(II) binding site suggested in [15]. An interesting feature in the crystallographic structure of lignin peroxidase [14] is a hydrogen-bond network comprising an imidazole nitrogen of the distal His-47 (cf. PcLG2 in Fig. 3), the amide group of Asn-84, and the peptide carbonyl of Glu-78. The presumed importance of this arrangement for enzymic catalysis is supported by the finding that the residues involved are evolutionary conserved, with His-47 and Asn-84 among the 10 invariant residues within the peroxidase superfamily [12]. In the sequence encoded by PG V, the Asn is replaced by a Set. Ser can also act as donor and acceptor in hydrogen bonds, and could possibly have a similar role as Asn in the proposed hydrogen bond network of the distal His. For that reason, the gene product of PG V may be an interesting species to study. This work was supported by grants from Swedish National Board for Technical Development and Swedish Council for Forestry and Agricultural Research, and by a fellowship to H.G. Becker from the Swedish Institute.
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L. Ji~nsson et al. / Biochimica et Biophysica Acta 1207 (1994) 255-259
PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLG1 TvPGV
MAFKSLIAFVA_LAAAVRAAP MAFGSLLAFV~ITRAAP MAFKQLFAAI TVALSLTAAN MAFKQLLAAL SVALTLQVTQ MAFKSLLSFV SVIGALQGAN MAFKTLLSIV SLLAAFQGAT MVSKFFTSLV S.LAAVLGAN MAFKTLASFV S~QVAS *
T ....... A V T A .... E S A V AAVVKEKRAT ~APNLDKRVA AA..LTRRVA ~A..LTRRVA ~S..LTRRVA ~A..LTRRVA
. . . . . . . . .
*----*
PcMNP1 PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TvPGV
QDLQETIFQN QDLQETLFQG DDIQId~MFHG DDIQQNLFHG EDLQQNLFHG DDLQENLFHG DDLQANLFDG DDIQKNLFDN
ee E.CGEDA/~EV D.CGED~HEV GQCGAEAHES GQCGAEAHEA GLCTAEAHES GLCTAEAHES GKCNAEAHES GECGEDVHES
PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TvPGV
LLFPTVEPNF LHFPTIEPNF MIFDTIETAF ITFSSIETTY AIFPEIETNF AIFSDIETAF TIFSHIETGF SIFASIETNF
o# • SANNGIDDSV NNLIPFMQKH SANSGIDDSV NNLLPFMQKH HPNIGLDEW AMQKPFVQKH HPNIGLDEWAIQKPFIAKH H P N I G L D E I I ELQKPFIAR_H H P N I G L D E I V ELQKPFIAd~H HPNIGLDEVV EKQRPFLQRH HASLGIDEIV GEQAPFIARH
- - - - * _ _ _ *
.
.
.
.
.
.
.
* _ *
.
.
.
## PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TvPGV
PGAPRLEFLA PGAPRLEFMA PGAPQMNFFT PGAPQMQFFL AGAPQLAAFV AGAPQLAAFV AGAPQLSAFV PGAPRLQFLL _ W * *
PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TVPGV
CPDGTR.VSH CPDGTR.VTN CANG.KTVGD CPDGVHTASN CPDGVNTATN CPDGVNTATN CPDGRHTATN CPDGVNTATN
.
# #+ IRLTFHDAIA IRLTFHDAIA IRLVFHDSIA LR~T~VFHDSIA LR~TFHDAIA LRLTFHDAIA LRLTFHDAIA LRLTFHDAIG
.
.
.
**------*
•
. . . . . .
AACCAFIPLA AACCAFIPLA ASCCAWFDVL AACCAWFPVL AACCQLFAVR AACCQLFAVR AACCALFPLR AACCQLFAVR * _ W *
. . . . . .
# + + ISRLQGPK.. . A G G G A D G S M ISQSLGPQ...~GGGADGSM ISPAMEAKGK FGGGGADGSI ISPKLQSQGK F~GGGADGSI ISPALEAQGI F~GGGADGSI ISPALEQQGI F~GGGADGSI ISPALEAQGK F~GGGADGSI FSRSAEANGT F~GGGADGSI
# NTISAADLVQ DTISAADLVQ G.VTPGDFIA G.VTRGDFIA N.ISVADFIQ N.LSVAD~IQ N.IGVAD~IQ N.LTVGD~IQ . . . . . .
* _ _ _
21 21 21 22 22 22 22 22
FAGAVALSNC FAGAVALSNC FAGAVALSNC FAGAVGVSNC FAGAIGASNC FAGAIGASNC FAGALGASNC FAGAVGVSNC
67 67 71 72 72 72 72 72
117 117 120 121 121 121 121 121
* * * * _ _ _ * * *
•
GRPNKTIAAV GRPNTTIPAV GRKPATQPAP GRPE~TQAAP GRKDATQPAP GRVDATQPAP GRKEPTRPAP GRPNATQPAP
DGLIPEPQDS VTKILQRFED EGLIPEPQDS VTKILQRFED DGLVPEPFHT VDQIIARVND DGLVPEPF~T IDQVLARMLD DGLVPEPF~T PDQIFDRLAD DGLVPEPFHT PDQIFARLAD DGLVPEPFHT PDQIFARIAD DKTIPEPFDT VDSILARFLD
A..GGFTPFE A..GNFSPFE A..GEFDELE A..GGFDEIE ASQGEFDPIL ASQGEFDEIL ASSGEFDEIL A..ADFSPAE
165 165 168 169 171 171 171 169
• ARADKVDQT I ARADKVDET I AAVNDVDPTV AAANDVDPTI AAANDVDPTK AAANDVDPTV AAANDVDPTV AA~EVDPT I
++ D A A P F D Sm T P F DAAPFDSTPF QGLPFDSTPG SGLPFDSTPG SGLPFDSTPE PGSPFDSTPE PGSPFDSTPE PGTPFDSTPE
LLKGVGFPGS LLKGTGFPGS QFRGTLFPGS QLRGTAFPGK QLRGTSFP~S LLNGTTFPGT QLKGTAFTGR QLRGTGFPGT
215 215 218 219 221 221 221 219
. . . . . .
# e#+ WSLLASHSV VVSLLASHTV LVWMLSAHSV TVWLLS~HSI TVWLLT~d~TV TVWLLVAHTV TVWLLTAHTI VVALLASHTI
+ TFDTQVFLEV TFDTQVFLEV IFDSQFFVET QFDSQFFVET LWDTQFFLET VWDTQFFVEV IFDSQFFLET LFDTQFFIET
Fig. 3. Alignment of peroxidase sequences from 7". versicolor (Tv) and P. chrysosporium (Pc). The sequences shown together with the translation product of PG V are manganese peroxidases encoded by MNP1 [10] and MP1 [20], lignin peroxidases from LG2 [19], LIP2 [9], LPGI [5], LPGII [7], and VLG1 [6] (translation product of VLG1 as in [7]). Residues invariant in the peroxidase superfamily (as defined in [12]) are marked by (#). Residues with side chains probably involved in the binding of Ca 2+ [14] are indicated by ( + ) . Potential N-glycosylation sites (NXS or NXT) are shown in bold face. Exon boundaries are indicated (double underscore of two residues = intron between codons, double underscore at one residue = split codon). An arrow marks the amino-terminus of mature proteins. Positions in which the PG V gene product should conform with invariant residues of manganese peroxidases (from MNP1 and MP1), but not with invariant residues of lignin peroxidases (from LG2, LIP2, LPGI, LPGII, and VLG1), are indicated by ( 0 ) .
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L. Ji~nsson et al. / Biochimica et Biophysica Acta 1207 (1994) 255-259
# PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TvPGV
~GEV~P ~GE~SP GGNQGE~SG TGIQGT~SP GGNQGE~SP GDNQG~ASP GPVQG~CP AGNQGE~SP . . . .
*--*
LPLGSGSDTG LPLGSGSDTG ~ ....... G L K ....... G ~ ....... G I A ....... G C A ....... G LP ....... G
. . . . . . . . . . . .
*
~RLQSDF~ ~RLQSDF~ EIRIQTDHTL ~RLQTDHLF ~RLQSDHTI EFRLQSDFAI EFRLQSDFAI ~RLQSDSEL *_*_*_*------
~DPRTACIW ~DERTAC~ ~DSRTACEW ~DSRTAC~ ~DSRTACEW ~DSRSACEW ~DQATACEW ~DSRTACEW * _ * _ _ _ * * _ *
QG~EQA~ QS~EQE~ QS~QSKL QS~QTKL QS~NQP~ QS~NQP~ QS~QT~ QS~QS~
265 265 261 262 264 264 264 262
* - - _ * _ _ * _ _ _
OQ
PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TvPGV
~ S F ~ S K ~ G H N ~ S LIDCSD~PV ~ S F ~ ~ILGHSRSS LIDCSD~PV ~DFQFIF~ LTQLGQDPNA~DCSDVIPL QEDFQFI~A LSTLGHD~A ~DCSEVIPA QQMFQ~FHD LSIFGQDINT L~CTE~PI Q~FQ~FHD L S I F G Q D I N S L ~ C T ~ P I1 QQMFQ~FHD LSILGQNIDD L~CT~IPI ~ A F ~ ~VIGQDVSQ LIDCS~IPM
PKPATGQ..P~FPASTGPQ PKPA~K..P ATFPATKGPK SKPIPGNGPF SFFPPGKSHS PKPV..NFGP SFFPAGKT~ P~PQ...GH THFPAGLSNA PAPLQ. . .GV T H F P A G L T ~ PRPLT...TR THFPA~HR PPPPA...SA ~FPAGLSNA
313 313 311 310 311 311 311 309
PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvL P G I I TvVLGI TvPGV
DLE.LSCPSE DLDTLTC~ DIEQ.A~T DIEQ.A~ST DIEQ.A~T DIDQ.PC~T DIEQ.A~LET D~Q.A~T
DGSMSCPGVQ ~GPA NGGMSCPGVQ FDGPA H~ ............ PPSPN .......... PP~K ......... P .............. KRV ............ S ..............
357 358 343 344 346 341 343 339
RFPTLTTQPG KFPTLTSDPG PFPSL~LPG PFPTLITAPG PFPTFPTDPG PFPTLPTDPG PFPTLPTDPG PFPTLQTDPG
ASQSLI~CP ATETLIPHCS PATSV~IPP PS~V~TPP PKTAVAP~PK PATSVAP~PL PRTGVAP~IP PETSVAP~PP
Fig. 3 (continued).
References [1] Eriksson, K.-E.L., Blanchette, R.A. and Ander, P. (1990) Microbial and Enzymatic Degradation of Wood and Wood Components, Springer, New York. [2] Evans, C.S., Gallagher, I.M., Atkey, P.T. and Wood, D.A. (1991) Biodegradation 2, 93-106. [3] Kirk, T.K., Tien, M., Kersten, P.J., Kalyanaraman, B., Hammel, K.E. and Farrell, R.L. (1990) Methods Enzymol. 188, 159-171. [4] Gold, M.H. and Alic, M. (1993) Microbiol. Rev. 57, 605-622. [5] Jfnsson, L. and Nyman, P.O. (1992) Biochimie 74, 177-183. [6] Black, A.K. and Reddy (1991) Biochem. Biophys. Res. Commun. 179, 428-435. [7] J6nsson, L. and Nyman, P.O. (1994) Biochim. Biophys. Acta, in press. [8] Johansson, T., Welinder, K.G. and Nyman, P.O. (1993) Arch. Biochem. Biophys. 300, 57-62. [9] Naidu, P.S., Zhang, Y.Z. and Reddy, C.A. (1989) EMBL Data Library Accession No. X15599. [10] Godfrey, B.J., Mayfield, M.B., Brown, J.A. and Gold, M.H. (1990) Gene, 93, 119-124. [11] Ballance, D.J. (1990) in Molecular Industrial Mycology, Systems
[12]
[13]
[14] [15]
[16] [17] [18]
[19] [20]
and Applications for Filamentous Fungi (Leong, S.A. and Berka, R.M., eds.), pp. 1-29, Marcel Dekker, New York. Welinder, K.G. and Gajhede, M. (1993) in Plant Peroxidases: Biochemistry and Physiology (Welinder, K.G., Rasmussen, S.K., Penel, C. and Greppin, H., eds.), pp. 35-42, University of Geneva. Veitch, N.C., Williams, R.J.P., Bray, R.C., Burke, J.F., Sanders, S.A., Thorneley, R.N.F. and Smith, A.T. (1992) Eur. J. Biochem. 207, 521-531. Poulos, T.L., Edwards, S.L., Wariishi, H. and Gold, M.H. (1993) J. Biol. Chem. 268, 4429-4440. Johnson, F., Loew, G.H. and Du, P. (1993) in Plant Peroxidases: Biochemistry and Physiology (Welinder, K.G., Rasmussen, S.K., Penel, C. and Greppin, H., eds.), pp. 31-34, University of Geneva. Banci, L., Bertini, I., Bini, T., Tien, M. and Turano, P. (1993) Biochemistry 32, 5825-5831. Henikoff, S. (1987) Methods Enzymol. 155, 156-165. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1993) Current Protocols in Molecular Biology, Wiley, New York. Ritch, T.G. and Gold, M.H. (1992) Gene 118, 73-80. Pease, E.A., Andrawis, A. and Tien, M. (1989) J. Biol. Chem. 264, 13531-13535.
Biochi~ic~a et B i o p h y s i c a A~ta
ELSEVIER
Biochimica et Biophysica Acta 1207 (1994) 255-259
Short Sequence-Paper
A novel type of peroxidase gene from the white-rot fungus Trametes versicolor 1 Leif J6nsson, Helen G. Becker
2, P e r O l o f N y m a n
*
Biochemistry, Chemical Center, University of Lund, P.O. Box 124, S-221 O0Lund, Sweden
Received 27 January 1994
Abstract
The wood-decaying fungus Trametes versicolor secretes a large number of peroxidase isozymes, presumed to partake in the degradation of lignin. From enzymic studies, two types of peroxidases have been distinguished: lignin peroxidases and manganese peroxidases. We here report the finding of a T. versicolor peroxidase gene, PG V, which displays several features not observed in previously studied peroxidase genes from white-rot fungi, such as a high number of introns (12). Eight of the 12 introns have positions equivalent to introns of peroxidase genes from another white-rot fungus, Phanerochaete chrysosporium. The gene structure of PG V appears to be primarily related to known lignin peroxidase genes, while the encoded mature 339-residue protein has several characteristics in common with manganese peroxidases. Analyses further indicate that PG V encodes a Ser instead of an Asn at a position regarded as invariant within the enzyme superfamily, with the side chain involved in hydrogen bonding with the distal His. Key words: Nucleotide sequence; Peroxidase gene; Lignin biodegradation; T. versicolor
The basidiomycete Trametes (Coriolus) versicolor has often been studied as an efficient degrader of wood [1,2]. T. versicolor secretes large amounts of a copper-containing phenoloxidase, laccase, which has been ascribed an important role in the ligninolytic action of the fungus. In addition, a large number of peroxidase isozymes have been shown to be secreted. In this regard, T. versicolor resembles another, extensively studied, white-rot fungus, Phanerochaete chrysosporium [3], which, however, does not produce any laccase [4]. The extracellular heme-containing peroxidases have been divided into two families, displaying differences in substrate specificity. Lignin peroxidases have been found to oxidize aromatic substrates, while manganese peroxidases have been suggested to act by oxidizing Mn(II) to Mn(III), which then in turn oxidizes phenolic lignin substructures. For both T. versicolor and P. chrysosporium, multiple genes have been shown to be a major source of the large
1The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number X77154. 2 Present address: Department of Chemical Enzymology, Chemical Faculty, M.V. Lomonosov Moscow University, Russia 117234. * Corresponding author. Fax: + 46 46 104534. 0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4838(94)00083-S
number of peroxidase isozymes observed. From P. chrysosporium, genes encoding lignin peroxidases and manganese peroxidases have been characterized (reviewed in [4]). From T. versicolor, several genes proposed to encode lignin peroxidases have been reported [5-7]. Of these sequences, the gene LPGI was found to encode a protein identical with an abundantly expressed lignin peroxidase isozyme, sequenced by Edman degradation of tryptic peptides to an extent of approximately 40% [8]. Analyses of amino-terminal ends of lignin peroxidases and manganese peroxidases isolated from T. versicolor indicate that a high degree of similarity between these enzymes can be expected [8]. Here, we report the finding of a T. versicolor peroxidase gene which displays unique features. The segment of DNA, the sequence of which is shown in Fig. 1, was obtained from a genomic library of T. versicolor, as described in [5]. The gene, P G V, is predicted to contain 12 introns. All previous peroxidase genes from T. versicolor contain six introns, identically positioned [7]. These six introns are also present in PG V as the three first and the three last introns (Fig. 2A). Of the remaining six introns in P G V, four have positions identical with LIP2 in Fig. 2A, which is the gene for a major lignin peroxidase isozyme (H2) in P. chrysosporium [3,9].
256
L. Ji~nsson et aL / Biochimica et Biophysica Acta 1207 (1994) 255-259
CTCGAGGTTGCCAAAA•GT•AGGA•ATC•AGTTCT•TGCCGGT•TCCCACTCATCGCA•AATGGCCTT•AAGACACTTGCGAGCTTCGTCTCTGT•CTTG MetAlaPheLysThrLeuAlaSerPheValSerValLeuA
i00
CCGCCCTCCAGGTCGC~AGCGGTCTGTTCTTCT~CTTCTCTACTCGTTTTCAACCTGCTGACATTGCACCAGGTGCC~TTAC~CGC~TCGCTTGCCC 200 laAlaLeuGlnValAlaSerG
lyAlaLeuThrArgArgValAlaCysPr
CGACGGCGTGAA•ACGGCTAC•AACGCGGCGTGCTGC•AGCTCTTCGC•GTCCGCGATGATAT•CAGAAGAAC•TGTTCGA•AACGGCGAGTGCGGTGAG
300
•AspG•yVa•AsnThrA•aThrAsnA•aA•aCys•ysG•nLeuPheA•aVa•ArgAspAspI•eG•nLysAsnLeuPheAspAsnG•yG•u•ysG•yG•u GACGTCCACGAGTCC~T~CGT~T~ACCTTCCACGA~GCCATTGGCTTCT~CCGCTCTGCGGAGC~TAA~GGCACCTTCGGGTAGGTCCTTGCACCTATCA AspVa•HisG•uSerLeuArgLeuThrPheHi•AspA•aI•eG•yPheSerArgSerA•aG•uA•aAsnG•yThrPheG•
400
CGACGT~GAAGCGTTGCTGAATC~TT~TCTTTACAC-CGGCGGAGGCGCGGA~C-C-CT~GATCTCCAT~TT~GCGTCCATCGAGACTAA~TTCCACGCGAGT yGlyGlyGlyAlaAspGlySerIleSerIlePheAlaSerIleGluThrAsnPheHisAlaSer
500
CTCGGTATCGA•GAGATCGTCGGCGAGCAGGCACCCTTCATTGC•CGC•A•AA•CT•A•CGTTGGCGACTTGTACGTCTTCTCCTGCCATACTTGGAAGT LeuGlyIleAspGluIleValGlyGluGlnAlaProPheIleAlaArgHisAsnLeuThrValGlyAspPh
600
TGTT•TAACTCGGAATCGTGCAGCATC•AGTTTGCTGGAGCCGT•GGTGTCAGCAACTGC•CCGGCGCCCCGCGCCTGCAGTTCCTGCTTGGTGAGTGGG eIleGlnPheAlaGlyAlaValGlyValSerAsnCysProGlyAlaProArgLeuGlnPheLeuLeuG
700
CAGGTCTCCGAATGTCTGGCTCTTTA•TGAACGC4•ATCTATCT•AGGC•GTCCCAACGCCA•T•AAC••GCGcCCGA•AAGACCATCCCGGAGCCATTCG lyArgProAsnAlaThrGlnProAlaProAspLysThrIleProGluProPheA
800
GTAAGAGATAAGAAACATTCCC-C~C~GTAAGGCGGTACTGACGCACTTTCAACACTAGACACCGT~GACTCCATTCTCGCCCGTTTCCTGGACGCCGCTG900 spThrValAspSerIleLeuAlaArgPheLeuAspAlaAlaA
ACTT~AC.C~CGC~GGAGGTCGTCGCTCTGCTGGCGTCGTATGTACTCTTCACGCTCGTCGGTTCACTCGATTACTGACCAGCACGTTTGTAGGCACACCA spPheSerProAlaGluValValAlaLeuLeuAlaSe rHisThrI
i000
T~C.~CTC-CGC-CCGATGAGGT~GA~GA~GATTC~CGG`a~k~TT~GAcT~CACGCCCGAGCTTTTCGACA~c~AGTTCTTCATCGAGA~T~AGCTGCGii00 •eA•aA•aA•aAspG•uVa•AspPr•ThrI•ePr•G•yThrPr•PheAspSerThrPr•G•uLeuPheAspThrG•nPhePheI•eG•uThrG•nLeuAr
CGGCACTGGGTT•CCTGGGTACGTTGTCGTTTGCTATGGCGCGAAACGTTACTGATGTAGAGCAGAACGGCCGGCAACCAGGGCGAGGTTCTGTCCCCTC qGlyThrGlyPheProGl yThrAlaGlyAsnGlnGlyGluValLeuSerProL
1200
TTCCCGGAGAGATGCGC•TGCAGTC•GACTCCGAGGTAAGATCAAGCTTCGGCCGTTGTGTTCTTGCACTCAACAAATCTTTTTCTAGCTCGCTCGTGAC euProGlyGluMetArgLeuGlnSerAspSerGlu LeuAlaArgAsp
1300
T~GGA~TGC~TC-CGAGTGC~AGTCCATGGTCAGTAAGTATTGCTCATTTGCTCTAT`a~kTCGACTATGTCCTGATTCATGTTTCCTTAGACAACCAGTC 1400 SerArgThrAlaCysGluTrpGlnSerMetValA
snAsnGlnSe
GAAGATGATGA~GGC~TTCGCGC~TGCCATGGCTAAGCTCGCGGTCATCGGCCAGGA~GTCAGCCAGTTGATTGACTGCTCCGAAGTGGTGCGTCCTCAC rLysMetMetThrA•aPheA•aA•aA•aMetA•aLysLeuA•aVa•I•eG•yG•nAspVa•SerG•nLeuI•eAspCysSerG•u•a•
1500
ACATGCCTTGCGTCT~TGACACGTTGAC~AATTTCTTCGCGAGTAGATCCCGATGCCC.C~GCC~CC-~GAGCGCCGCGCACTTCCCCGCCGGACTTAGC IleProMetProProProProAlaSerAlaAlaHisPheProAlaGlyLeuSer
1600
AACGCGGACGTCGAGCAGGCTGTGCGTGTTTCGATGTTGATGGTCTTCCCCATGTTGCTCACGTATGCCGATATAGTGCGCCGAGACCCCCTTCCCGACT AsnAlaAspValGluGlnAla CysAlaGluThrProPheProThr
1700
CTC•AGA•CGAC•••GGA••CGAGACCTCC-GTGC.CCCCTGTGTAAGTGA•T•CAC-GGCTATCTCTCACCTGCTAGGCTCTAACTCTTGGATCTCTCTCTA LeuGlnThrAspProGlyProGluThrSerValAlaProVa
1800
GCCCCCC GTCTTAAC-CTAAGCACAGGAGATGTCGGCGGAAC-CTCTGC TATAGCGAGT GC TT TGCT TATTTAGAAATGGTCGTT TGT TACTAT IProProSerEND
1892
Fig. 1. The nucleotide sequence of PG V. The DNA segment shown is from a 3 kbp XhoI-fragment which was inserted into pGEM-TZf( + ). The segment shown was sequenced in one direction using a series of nested deletions [17], and in the other direction by the primer synthesis approach. Standard methods were employed for cloning and dideoxy-sequencing [18]. Computer analyses of sequences were performed using the GCG programs (Version 7, 1991, Genetics Computer Group, Inc., Madison, Wisconsin, USA). The predicted translation product is indicated in the figure. The sequence has been submitted to the EMBL/GenBank Database (accession number X77154).
L. Jfnsson et al. / Biochimica et Biophysica Acta 1207 (1994) 255-259
As previously shown [5], four of the six introns in LPGI are positioned identically with introns of P. chrysosporium peroxidase genes. The corresponding figures for PG V are eight introns in common out of twelve (see Fig. 2A). Fig. 2B shows that the intron positions in PG V differ considerably from those reported for a manganese peroxidase gene [10]. Thus, on the gene level, PG V appears related to lignin peroxidases. The translation initiation environment of PG V (CACAATGGC) is in full agreement with the consensus for filamentous fungi [11] (CAMMATGNC, where M = A or C), indicating that PG V may be expressed. The gene is predicted to encode a 339-residue mature protein, preceded by 26 residues. These residues are proposed to be removed in two proteolytic steps, the first accomplished by a signal peptidase and the second by a Kex2-related endopeptidase. Such an arrangement conforms with previously studied lignin peroxidase genes [4,7], while the manganese peroxidase genes from P. chrysosporium, so far studied, do not encode the two basic residues of the second proteolytic cleavage site. Previously known T. versicolor peroxidase sequences are clearly more similar to lignin peroxidases from P. chrysosporium (over 60% identity between encoded mature proteins) than to manganese peroxidases (47-50% identity). PG V, however, does not follow this pattern. The identity with P. chrysosporium lignin peroxidases is roughly the same as with manganese peroxidases. P. chrysosporium manganese peroxidases display a stronger similarity to the translation product of PG V (56-57%) than to lignin peroxidases (50% or less, even within P. chrysosporium). In Fig. 3, it can be seen that for several positions conserved in lignin peroxidases, PG V instead conforms with manganese peroxidases. The most significant difference in amino acid composi-
A. LPGI PGV
LI Pfl LG2
B° /:~V
I
I
I I
~ I
.~NP1 I I I I Fig. 2. Comparison of intron positions. In (A) PG V is compared with LPGI (lignin peroxidase gene from T. versicolor), LIP2 [9] and LG2 [19] (lignin peroxidase genes from P. chrysosporium). In (B) PG V is compared with MNP1 [10] (a manganese peroxidase gene from P. chrysosporium ).
257
tion between T. versicolor lignin and manganese peroxidases has been reported to be the content of Ser [8]. The high content of Ser (24 residues) in the mature protein coded for by PG V is consistent with the reported value for manganese peroxidases (approx. 24 Ser, compared with 16 for lignin peroxidases). The amino-terminal sequence encoded by PG V is also more similar to manganese peroxidases isolated from T. versicolor than to lignin peroxidases. Of the 35 terminal residues reported for manganese peroxidase isozyme TvMP2 [8], only 2 are mismatches when compared with the sequence encoded by PG V. Preliminary studies of a presumed gene for isozyme TvMP2, isolated in our laboratory (Johansson, T., personal communication), indicate that a high degree of identity is not restricted to the amino-terminal portion. Residues interacting with reducing substrates have not been conclusively identified for either lignin or manganese peroxidases. Two Phe in horseradish peroxidase, a structurally related peroxidase [12], have been shown to interact with aromatic substrates [13]. The corresponding residues in a translation product of PG V would be 149 and 150. In PG V, one of these residues, a Phe, is identical with lignin peroxidases, while the other one, an Asp, is identical with manganese peroxidases (Fig. 3). Based on model building studies, residues interacting with an aromatic substrate have been suggested for lignin peroxidase [14] (corresponding to residues 82 (His), 85 (Ile), 148 (Phe), 184 (Val), and 222 (Gln) from PcLG2 in Fig. 3). Residues in manganese peroxidase that may interact with Mn(II) have also been suggested [15,16] (corresponding to residues 35 (Glu), 39 (Glu), and 179 (Asp) [15] or 84 (Asp), 85 (Asp), 179 (Asp), and 182 (Asp) [16] of the manganese peroxidase sequences in Fig. 3). The residues encoded by PG V do not show any conspicuous deviations from the substrate binding sites proposed for either lignin peroxidase or manganese peroxidase, and are fully consistent with the Mn(II) binding site suggested in [15]. An interesting feature in the crystallographic structure of lignin peroxidase [14] is a hydrogen-bond network comprising an imidazole nitrogen of the distal His-47 (cf. PcLG2 in Fig. 3), the amide group of Asn-84, and the peptide carbonyl of Glu-78. The presumed importance of this arrangement for enzymic catalysis is supported by the finding that the residues involved are evolutionary conserved, with His-47 and Asn-84 among the 10 invariant residues within the peroxidase superfamily [12]. In the sequence encoded by PG V, the Asn is replaced by a Set. Ser can also act as donor and acceptor in hydrogen bonds, and could possibly have a similar role as Asn in the proposed hydrogen bond network of the distal His. For that reason, the gene product of PG V may be an interesting species to study. This work was supported by grants from Swedish National Board for Technical Development and Swedish Council for Forestry and Agricultural Research, and by a fellowship to H.G. Becker from the Swedish Institute.
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PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLG1 TvPGV
MAFKSLIAFVA_LAAAVRAAP MAFGSLLAFV~ITRAAP MAFKQLFAAI TVALSLTAAN MAFKQLLAAL SVALTLQVTQ MAFKSLLSFV SVIGALQGAN MAFKTLLSIV SLLAAFQGAT MVSKFFTSLV S.LAAVLGAN MAFKTLASFV S~QVAS *
T ....... A V T A .... E S A V AAVVKEKRAT ~APNLDKRVA AA..LTRRVA ~A..LTRRVA ~S..LTRRVA ~A..LTRRVA
. . . . . . . . .
*----*
PcMNP1 PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TvPGV
QDLQETIFQN QDLQETLFQG DDIQId~MFHG DDIQQNLFHG EDLQQNLFHG DDLQENLFHG DDLQANLFDG DDIQKNLFDN
ee E.CGEDA/~EV D.CGED~HEV GQCGAEAHES GQCGAEAHEA GLCTAEAHES GLCTAEAHES GKCNAEAHES GECGEDVHES
PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TvPGV
LLFPTVEPNF LHFPTIEPNF MIFDTIETAF ITFSSIETTY AIFPEIETNF AIFSDIETAF TIFSHIETGF SIFASIETNF
o# • SANNGIDDSV NNLIPFMQKH SANSGIDDSV NNLLPFMQKH HPNIGLDEW AMQKPFVQKH HPNIGLDEWAIQKPFIAKH H P N I G L D E I I ELQKPFIAR_H H P N I G L D E I V ELQKPFIAd~H HPNIGLDEVV EKQRPFLQRH HASLGIDEIV GEQAPFIARH
- - - - * _ _ _ *
.
.
.
.
.
.
.
* _ *
.
.
.
## PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TvPGV
PGAPRLEFLA PGAPRLEFMA PGAPQMNFFT PGAPQMQFFL AGAPQLAAFV AGAPQLAAFV AGAPQLSAFV PGAPRLQFLL _ W * *
PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TVPGV
CPDGTR.VSH CPDGTR.VTN CANG.KTVGD CPDGVHTASN CPDGVNTATN CPDGVNTATN CPDGRHTATN CPDGVNTATN
.
# #+ IRLTFHDAIA IRLTFHDAIA IRLVFHDSIA LR~T~VFHDSIA LR~TFHDAIA LRLTFHDAIA LRLTFHDAIA LRLTFHDAIG
.
.
.
**------*
•
. . . . . .
AACCAFIPLA AACCAFIPLA ASCCAWFDVL AACCAWFPVL AACCQLFAVR AACCQLFAVR AACCALFPLR AACCQLFAVR * _ W *
. . . . . .
# + + ISRLQGPK.. . A G G G A D G S M ISQSLGPQ...~GGGADGSM ISPAMEAKGK FGGGGADGSI ISPKLQSQGK F~GGGADGSI ISPALEAQGI F~GGGADGSI ISPALEQQGI F~GGGADGSI ISPALEAQGK F~GGGADGSI FSRSAEANGT F~GGGADGSI
# NTISAADLVQ DTISAADLVQ G.VTPGDFIA G.VTRGDFIA N.ISVADFIQ N.LSVAD~IQ N.IGVAD~IQ N.LTVGD~IQ . . . . . .
* _ _ _
21 21 21 22 22 22 22 22
FAGAVALSNC FAGAVALSNC FAGAVALSNC FAGAVGVSNC FAGAIGASNC FAGAIGASNC FAGALGASNC FAGAVGVSNC
67 67 71 72 72 72 72 72
117 117 120 121 121 121 121 121
* * * * _ _ _ * * *
•
GRPNKTIAAV GRPNTTIPAV GRKPATQPAP GRPE~TQAAP GRKDATQPAP GRVDATQPAP GRKEPTRPAP GRPNATQPAP
DGLIPEPQDS VTKILQRFED EGLIPEPQDS VTKILQRFED DGLVPEPFHT VDQIIARVND DGLVPEPF~T IDQVLARMLD DGLVPEPF~T PDQIFDRLAD DGLVPEPFHT PDQIFARLAD DGLVPEPFHT PDQIFARIAD DKTIPEPFDT VDSILARFLD
A..GGFTPFE A..GNFSPFE A..GEFDELE A..GGFDEIE ASQGEFDPIL ASQGEFDEIL ASSGEFDEIL A..ADFSPAE
165 165 168 169 171 171 171 169
• ARADKVDQT I ARADKVDET I AAVNDVDPTV AAANDVDPTI AAANDVDPTK AAANDVDPTV AAANDVDPTV AA~EVDPT I
++ D A A P F D Sm T P F DAAPFDSTPF QGLPFDSTPG SGLPFDSTPG SGLPFDSTPE PGSPFDSTPE PGSPFDSTPE PGTPFDSTPE
LLKGVGFPGS LLKGTGFPGS QFRGTLFPGS QLRGTAFPGK QLRGTSFP~S LLNGTTFPGT QLKGTAFTGR QLRGTGFPGT
215 215 218 219 221 221 221 219
. . . . . .
# e#+ WSLLASHSV VVSLLASHTV LVWMLSAHSV TVWLLS~HSI TVWLLT~d~TV TVWLLVAHTV TVWLLTAHTI VVALLASHTI
+ TFDTQVFLEV TFDTQVFLEV IFDSQFFVET QFDSQFFVET LWDTQFFLET VWDTQFFVEV IFDSQFFLET LFDTQFFIET
Fig. 3. Alignment of peroxidase sequences from 7". versicolor (Tv) and P. chrysosporium (Pc). The sequences shown together with the translation product of PG V are manganese peroxidases encoded by MNP1 [10] and MP1 [20], lignin peroxidases from LG2 [19], LIP2 [9], LPGI [5], LPGII [7], and VLG1 [6] (translation product of VLG1 as in [7]). Residues invariant in the peroxidase superfamily (as defined in [12]) are marked by (#). Residues with side chains probably involved in the binding of Ca 2+ [14] are indicated by ( + ) . Potential N-glycosylation sites (NXS or NXT) are shown in bold face. Exon boundaries are indicated (double underscore of two residues = intron between codons, double underscore at one residue = split codon). An arrow marks the amino-terminus of mature proteins. Positions in which the PG V gene product should conform with invariant residues of manganese peroxidases (from MNP1 and MP1), but not with invariant residues of lignin peroxidases (from LG2, LIP2, LPGI, LPGII, and VLG1), are indicated by ( 0 ) .
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# PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TvPGV
~GEV~P ~GE~SP GGNQGE~SG TGIQGT~SP GGNQGE~SP GDNQG~ASP GPVQG~CP AGNQGE~SP . . . .
*--*
LPLGSGSDTG LPLGSGSDTG ~ ....... G L K ....... G ~ ....... G I A ....... G C A ....... G LP ....... G
. . . . . . . . . . . .
*
~RLQSDF~ ~RLQSDF~ EIRIQTDHTL ~RLQTDHLF ~RLQSDHTI EFRLQSDFAI EFRLQSDFAI ~RLQSDSEL *_*_*_*------
~DPRTACIW ~DERTAC~ ~DSRTACEW ~DSRTAC~ ~DSRTACEW ~DSRSACEW ~DQATACEW ~DSRTACEW * _ * _ _ _ * * _ *
QG~EQA~ QS~EQE~ QS~QSKL QS~QTKL QS~NQP~ QS~NQP~ QS~QT~ QS~QS~
265 265 261 262 264 264 264 262
* - - _ * _ _ * _ _ _
OQ
PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvLPGII TvVLGI TvPGV
~ S F ~ S K ~ G H N ~ S LIDCSD~PV ~ S F ~ ~ILGHSRSS LIDCSD~PV ~DFQFIF~ LTQLGQDPNA~DCSDVIPL QEDFQFI~A LSTLGHD~A ~DCSEVIPA QQMFQ~FHD LSIFGQDINT L~CTE~PI Q~FQ~FHD L S I F G Q D I N S L ~ C T ~ P I1 QQMFQ~FHD LSILGQNIDD L~CT~IPI ~ A F ~ ~VIGQDVSQ LIDCS~IPM
PKPATGQ..P~FPASTGPQ PKPA~K..P ATFPATKGPK SKPIPGNGPF SFFPPGKSHS PKPV..NFGP SFFPAGKT~ P~PQ...GH THFPAGLSNA PAPLQ. . .GV T H F P A G L T ~ PRPLT...TR THFPA~HR PPPPA...SA ~FPAGLSNA
313 313 311 310 311 311 311 309
PcMNPI PcMPI PcLG2 PcLIP2 TvLPGI TvL P G I I TvVLGI TvPGV
DLE.LSCPSE DLDTLTC~ DIEQ.A~T DIEQ.A~ST DIEQ.A~T DIDQ.PC~T DIEQ.A~LET D~Q.A~T
DGSMSCPGVQ ~GPA NGGMSCPGVQ FDGPA H~ ............ PPSPN .......... PP~K ......... P .............. KRV ............ S ..............
357 358 343 344 346 341 343 339
RFPTLTTQPG KFPTLTSDPG PFPSL~LPG PFPTLITAPG PFPTFPTDPG PFPTLPTDPG PFPTLPTDPG PFPTLQTDPG
ASQSLI~CP ATETLIPHCS PATSV~IPP PS~V~TPP PKTAVAP~PK PATSVAP~PL PRTGVAP~IP PETSVAP~PP
Fig. 3 (continued).
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[19] [20]
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