Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana Karen G. Welinder 1,2 , Annemarie F. Justesen 1 , Inger V. H. Kjærsga ˚ rd 1 , Rikke B. Jensen 1 , Søren K. Rasmussen 3 , Hans M. Jespersen 1 and Laurent Duroux 2 1 Department of Protein Chemistry, University of Copenhagen, Denmark; 2 Department of Biotechnology, Aalborg University, Denmark; 3 Plant Genetics, Risø National Laboratory, Denmark Understanding peroxidase function in plants is complicated by the lack of substrate specificity, the high number of genes, their diversity in structure and our limited knowledge of peroxidase gene transcription and translation. In the present study we sequenced expressed sequence tags (ESTs) enco- ding novel heme-containing class III peroxidases from Arabidopsis thaliana and annotated 73 full-length genes identified in the genome. In total, transcripts of 58 of these genes have now been observed. The expression of individual peroxidase genes was assessed in organ-specific EST libraries and compared to the expression of 33 peroxidase genes which we analyzed in whole plants 3, 6, 15, 35 and 59 days after sowing. Expression was assessed in root, rosette leaf, stem, cauline leaf, flower bud and cell culture tissues using the gene-specific and highly sensitive reverse transcriptase- polymerase chain reaction (RT-PCR).We predicted that 71 genes could yield stable proteins folded similarly to horse- radish peroxidase (HRP). The putative mature peroxidases derived from these genes showed 28–94% amino acid sequence identity and were all targeted to the endoplasmic reticulum by N-terminal signal peptides. In 20 peroxidases these signal peptides were followed by various N-terminal extensions of unknown function which are not present in HRP. Ten peroxidases showed a C-terminal extension indicating vacuolar targeting. We found that the majority of peroxidase genes were expressed in root. In total, class III peroxidases accounted for an impressive 2.2% of root ESTs. Rather few peroxidases showed organ specificity. Most importantly, genes expressed constitutively in all organs and genes with a preference for root represented structurally diverse peroxidases (< 70% sequence identity). Further- more, genes appearing in tandem showed distinct express- ion profiles. The alignment of 73 Arabidopsis peroxidase sequences provides an easy access to the identification of orthologous peroxidases in other plant species and will provide a common platform for combining knowledge of peroxidase structure and function relationships obtained in various species. Keywords: EST; expression analysis by RT-PCR; peroxi- dase gene annotation; peroxidase structure; propeptides. Peroxidase enzymes have challenged chemists and biologists for more than 70 years and have been used in a great number of analytical applications [1]. The majority of peroxidases contain an extractable heme (Fe 3+ protopor- phyrin IX) center, whereas others contain a cytochrome c type heme, a selenium center or a vanadium center. Peroxidases react first with a peroxide to yield highly oxidizing intermediates with redox potentials up to 1000 mV and thereafter with a variety of organic or inorganic reducing substrates, which are often oxidized to form radicals. Peroxidase activity was detected early in horseradish roots (reviewed in [1]), which is still the major source of commercial heme peroxidases. In addition, peroxidases have been isolated from a variety of plant, animal, fungal and bacterial sources. The bacterium Escherichia coli expresses a single intracellular heme peroxi- dase with dual catalase–peroxidase activities [2], a finding confirmed by its genome sequence [3]. Mitochondrial yeast cytochrome c peroxidase, chloroplast and cytosol plant ascorbate peroxidases are rather similar in amino acid sequence to the bacterial enzymes, and they are collectively referred to as class I peroxidases [4]. These intracellular peroxidases appear to function as protective peroxide scavengers and they constitute in plants a small family of 7–10 genes, encoding both soluble and membrane bound enzymes [5]. White-rot fungi like Phanerochaete chrysospo- rium and Trametes versicolor contain a small gene family encoding approximately 10 different lignin-degrading or Mn-dependent heme peroxidases. In contrast, the ink cap fungus Coprinus cinereus contains only a single peroxidase gene [6,7]. The extracellular fungal peroxidases (class II) can participate in secondary metabolism under conditions of limited nutritional supply [8]. The classical plant peroxidases (class III) are targeted via the endoplasmic reticulum (ER) to the outside of the plant cell or to the vacuole. They are Correspondence to K. G. Welinder, Department of Biotechnology, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark. Fax: + 45 98141808, Tel.: + 45 96358467, E-mail: welinder@bio.auc.dk Abbreviations: AtP, transcribed A. thaliana (class III) peroxidase; BP, barley peroxidase; dbEST, database of ESTs; ef-1a, elongation factor-1a; EST, expressed sequence tag; HRP, horseradish peroxidase; SBP, soybean peroxidase; TC, tentative consensus. Notes: Equal contributions were made to this work by A. F. J., L. D. and H. M. J. The GenBank accession numbers for the nucleotide sequence data produced are listed in Table 1. (Received 19 August 2002, revised 8 October 2002, accepted 15 October 2002) Eur. J. Biochem. 269, 6063–6081 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03311.x ascribed a variety of functional roles in plant biology, which include lignification, suberization, auxin catabolism, def- ense, stress and developmentally related processes (reviewed in [9,10]). Prior to the present study it was known that horseradish contained at least nine different genes for class III peroxidases [11]. With this background, it seemed ideal to study the entire repertory of plant peroxidase genes in the model plant Arabidopsis thaliana,which belongs to the same botanical family, taking advantage of the expressed sequence tag (EST) sequencing programs in progress [12–14], as well as the results of the Arabidopsis genomic sequencing project [15]. Here we report the complete sequencing and mRNA expression analyses of class III Arabidopsis peroxidase transcripts mostly obtained from the EST projects, and the predicted protein structures derived from all 73 Arabidopsis peroxi- dase genes [16]. MATERIALS AND METHODS DNA sequencing and gene annotation BLAST and Entrez services at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) [17,18] were used to search databases (nonredundant and dbEST). EST clones were obtained from the Arabidopsis Biological Resource Center, Ohio State University [12,13], Genome Systems (Genome Systems Inc, St Louis, USA), and the Kasuza Institute [14]. Plasmid DNA purification and sequencing were performed as described previously [19] and both strands were sequenced. Genes encoding class III peroxidases in Arabidopsis were searched for in the Munich Information Center for Protein Sequences (MIPS) [20] and The Institute for Genomic Research (TIGR) [21] annotated databases using the keyword ÔperoxidaseÕ. Lists of genes were extracted and those coding for class I peroxidases (ascorbate peroxidases), glutathione peroxidases and catalases were removed, leaving a set of 75 nonredundant acces- sions. Predictions of intron splice-sites were done with NETPLANTGENE [22] (http://www.cbs.dtu.dk/services/). Putative transcriptional start sites and TATA-like boxes were mapped in the 5¢-UTR with the eukaryotic neural net- work promoter prediction server at http://www.fruitfly. org/seq_tools/promoter.html, using human and fruit-fly data. Predicted results were compared with known 5¢-UTRs from publicly available cDNA sequences. Nuc- leotide compositions of the 5¢-UTRs were computed as described in [23]. Protein sequence alignment Amino acid sequences were derived from the coding regions of the expressed genes using the program NETSTART for plants [24] (http://www.cbs.dtu.dk/services/NetStart/) for predicting initiating Met. The N-terminal signal peptides were predicted with the SIGNALP program [25] (http:// www.cbs.dtu.dk/services/SignalP-2.0/) and checked with the TARGETP program [26] (http://www.cbs.dtu.dk/services/ TargetP/). The alignments were performed with the CLUSTALX program [27] using the GONNET substitution matrices [28] on truncated sequences corresponding to residues 1–305 of mature HRPC. A first alignment was done with all sequences to obtain similarity clusters. An improved alignment was built using the profile alignment mode of CLUSTALX . First, a group of sequences highly similar to horseradish peroxidase C (HRPC) was aligned taking into account the secondary structure assignments for HRPC (default settings in CLUSTALX ). This group of aligned sequences was then used as a core onto which clusters of sequences were added sequentially. Finally, minor manual adjustments were made to exclude an excessive number of gaps. In calculating the pairwise distances, the sequence length was defined as all matched residues, not counting gaps. Calculation of pairwise distances and isoelectric points used only aligned full-length sequences, which were trun- cated to start at the position corresponding to the N-terminal pyroglutamate residue of mature HRPC, and ending at the position corresponding to HRPC residue N305 [29]. Plant material and RNA purification A. thaliana seeds, ecotype Columbia were kindly provided by F. Floto, and cell suspension culture by O. Mattsson, both at the Department of Plant Physiology, University of Copenhagen. Plants were grown in plastic containers on Murashige and Skoog medium (catalog no. 2606, Betatech) at 25 °C, 16 h light (3000 lux). Plants were harvested 3, 6, 15, 35 and 59 days after sowing. Plants older than 15 days were dissected into roots, rosettes, cauline leaves, stems and flower buds and the tissue was transferred immediately into liquid nitrogen and ground in a mortar. Total RNA was isolated using an RNeasy total RNA purification kit (QIAGEN) according to the manufacturer’s instructions. The quality of the RNA was evaluated by gel electrophor- esis and by measuring A 260 /A 280 . Purified RNA was stored at )80 °C. RT-PCR analysis The RT-PCR analyses were performed using the Perkin- Elmer GeneAmpÒ RNA PCR kit. An oligo(d[T] 16 ) primer was used for the first strand synthesis. Primers specific to each peroxidase gene were used for the second strand synthesis and PCR amplification (Supplementary material, Table S1). The specificity of each set of primers was optimized using the corresponding cDNA clone. Different combinations of annealing temperatures (60– 65 °C) and concentrations of MgCl 2 (1.0–2.0 m M )were tested to find the optimal conditions at which the primers were specific. When possible, the primers were designed to anneal in the 5¢ sequence encoding the signal peptide or in the 3¢-UTR. Primer sets were tested for specificity in a PCR, performed on a mixture of cDNA clones encoding all the peroxidases investigated, including and excluding the clone encoding the peroxidase for which the primers were designed. RT-PCR analyses were performed twice for each peroxidase using two different reverse transcribed reactions for each time point and organ. As a control of the quality of the mRNA, RT-PCR was performed with primers specific for the elongation factor-1a (ef-1a)[19]. The RT-PCR products were analyzed on a 1% (w/v) agarose gel. 6064 K. G. Welinder et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Digital expression analysis Transcription profiles were inferred from peroxidase EST counts, abstracted from TIGR A. thaliana Gene Index [30] (AtGI release version 6, May 2001) using ÔperoxidaseÕ as a keyword for the search. Each Tentative Consensus (TC) accession was verified and assigned to a unique peroxidase gene [15,20]. For each accession, the number of ESTs per library was counted. EST libraries (TIGR codes indicated by ¢#¢) were grouped according to organ: 1, root Columbia, #5336 [14], root-1 and -2 Col0 Columbia, #2336 and #2337 (Genome Systems, Inc.); 2, seedling hypocotyl CD4-13, -14, -15 and -16, #NH28, #NH25, #NH26 and #NH27 [12]; 3, rosette-1, -2 and -3 Col0 Columbia, #2338, #2340 and #2341 (Genome Systems, Inc.); 4, above-ground organs two to six weeks-old, #4063, #5335 and #3792 [14], Ors-A green shoot, #NH12, shoot 2-weeks old, #NH29; 5, flower bud Columbia, #5337 [14], inflorescence-1 and -2 Col0 Colum- bia, #2334 and #2335 (Genome Systems, Inc.), flower bud Grenoble-A and -B, #NH08 and #NH09, inflorescence young flower CD4-6, #NH36; 6, green silique Columbia, #5339 [14], green silique GIF-Seed A, A + B and GIF- Silique B, #NH05, #NH06 and #NH07, immature siliques, #2369; 7, developing seeds, #5564 [31], early developing seeds, #5576, germinating seed, #2370; 8, whole seedling Versailles-VB, -VC and -VD, #NH18, #NH19 and #NH20; 9, various, consisting mainly of the mixed organs k-PRL2 library, #NH11 contributing 27 631 ESTs [12] as well as all remaining EST libraries used in TCs by TIGR: #NH10, #2339, #2342, #4924, #NH03, #NH39, #4921, #4932, #5338, #NH02, #NH01, #NH13, #NH30, #6523, #6524, #7052, #7053, #7054, #7055, #1725, #2373, #2741, #NH04, #NH14, #NH15, #NH16, #NH17, #NH35, #NH44, #NH31, #NH32, #NH34, #NH37, #NH38, #NH40, #NH41, #NH43. RESULTS AND DISCUSSION cDNA and gene sequences The total number of ESTs from Arabidopsis has recently increased to 111 206, including 942 class III peroxidase clones (TIGR release v 6.0), or 0.85% of the total. Genes encoding class III peroxidases are easily identified by the most conserved active site motif (Fig. 1), which is located approximately 70 amino acids from the initiating Met residue, or 210 nucleotides from the initiating AUG codon. The selected clones were sequenced completely on both strands and the putative peroxidases called AtP1 to AtP38. The sequences have been deposited at GenBank or EMBL databases under the accession numbers listed in Table 1. Additional sequences of Arabidopsis peroxidase transcripts were obtained from the literature and our own work, AtPCa, -Cb, -Ea, -N, -A2, -RC (original names retained, except for RCIIIa). Recent large-scale Arabidopsis cDNA sequencing by the Riken Genomic Sciences Center, Yoko- hama, Japan, and Ceres Inc., Malibu, California, has currently brought the total of nonredundant peroxidase transcripts up to 57, AtP39 to AtP51. These 57 transcripts represent 58 genes, as two identical genes are represented by AtP11 (Fig. 1; Table 1). The MIPS gene names are used for the peroxidase genes for which no transcripts have been observedsofar. Analysis of the Arabidopsis genome [15] revealed a total of 73 full-length class III peroxidase genes, two pseudo- genes, and six fragments spread rather evenly on the five Arabidopsis chromosomes[16;L.DurouxandK.G. Welinder, unpublished observations]. Introns were localized and their phase determined. Results are reported in Table 1, and intron locations mapped to the protein sequences in Fig. 1 (highlighted in reverse print). Introns 1, 2 and 3 are predominant. The peroxidase-encoding DNA sequences have been analyzed thoroughly and annotated as in [23]. Table 1 provides an overview of all peroxidase genes and their introns, the percentage adenine content of 5¢-UTRs, predicted initiating Met, lengths of preproperoxidases and ER-signal peptides, and calculated isoelectric points of the putative mature polypeptides truncated to HRPC positions 1–305. The protein sequences predicted from the 73 genes are aligned in Fig. 1 as a base for the comprehensive structural characterization of the entire class III peroxidase repertory of a flowering plant. Sites of initiating Met and ER-signal cleavage were predicted using both hidden Markov (scores reported in Table 1) and neural network methods. Possible alternative sites are shown in Supple- mentary material, Fig. S1. The nucleotide sequences, anno- tation and percentage nucleotides of 5¢-UTRs of 73 peroxidase genes are given in the Supplementary material accompanying this paper (Fig. S2, and Table S2). Nucleotide differences have been observed between similar cDNA clones, and between cDNA and the corres- ponding gene. This can be ascribed to either allelic variations or to different ecotypes despite the fact that all were designated Columbia. Kjærsga ˚ rd et al. [19] described Fig. 1. Alignment of the amino acid sequences of putative mature per- oxidases predicted from the 73 class III Arabidop sis peroxidase genes. The 58 transcribed genes are referred to by AtP# names; the rest by MIPS gene numbers. The sequences are sorted according to similarity, and peroxidases > 70% amino acid identity are boxed, alternating in blue and grey. The Arabidopsis peroxidases are compared to horse- radish peroxidase HRPC. The a-helices, A–J, observed in HRPC (top), and residue or position numbers also refer to HRPC. Conserved res- idues (bottom) include invariant (uppercase), and highly conserved (lowercase). Active site residues are in red; side chain ligands to the distal and proximal Ca 2+ ions are in blue; cysteine residues involved in disulfide bridges 11–91, 44–49, 97–301 and 177–209 are in yellow; an invariant ion-pair motif are on a grey background; and putative N-glycosylated triplets are in green. Unusual residues are highlighted on a yellow background. Residue 1 (Z) in HRPC is pyroglutamate, a modification that is likely for all AtPs starting with glutamine (Q). Predicted N-terminal ER-targeting signals have been removed (Table 1; Supplementary material, Fig. S1) with alternative predic- tions for AtP32 and AtP1 indicated in brackets. Some AtPs show N-terminal extensions relative to HRPC residue 1, referred to as NX propeptides in the text. C-terminal extensions, CX propeptides, are shown in italics, and are not thought to be present in mature peroxi- dase. Intron positions in the corresponding genes are indicated by residues in reversed print, phase 0 introns between two marked resi- dues, phase 1 and 2 introns within a single residue. Two genes marked by (?) are unlikely to form stable proteins. At4g16270 ? encodes a 21-residue insert after intron 1 at HRPC position 48. At4g33870 ? has an unusual intron 2 at position 122, and an extra intron at position 236, both of which give rise to abnormal sequences (marked in yellow). Ó FEBS 2002 73 peroxidases from Arabidopsis (Eur. J. Biochem. 269) 6065 twosetsofcDNAsforAtP1,AtP1aandAtP1b,withthree conserved nucleotide mismatches, and two sets for AtP2, AtP2a and AtP2b, with 19 mismatches and three deletions. AtP1b and AtP2a are identical in sequence to the genes At4g21960 and At2g37130, respectively. The nucleotide differences result in one amino acid substitution within the 6066 K. G. Welinder et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 1. (Continued). Ó FEBS 2002 73 peroxidases from Arabidopsis (Eur. J. Biochem. 269) 6067 Fig. 1. (Continued). 6068 K. G. Welinder et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 1. (Continued). Ó FEBS 2002 73 peroxidases from Arabidopsis (Eur. J. Biochem. 269) 6069 Fig. 1. (Continued). 6070 K. G. Welinder et al.(Eur. J. Biochem. 269) Ó FEBS 2002 putative mature AtP1, and three in AtP2. Differences between transcripts and corresponding genes for AtP4, AtP5, AtP7 and AtPN gave rise to one amino acid substitution within the mature proteins. Two substitutions were found for AtPCb and AtP6, and six for AtP14. Other observed differences resulted from splice variants, for exam- ple in AtP9, AtP15 [32], AtP36 (GenBank AF451952) and AtPEa (TIGR TC115446 and TC115444). Protein structure of 73 putative peroxidases Figure 1 shows Arabidopsis peroxidases without their predicted ER-signal peptides, sorted and aligned according to similarity. The same similarity order is adopted in Tables 1 and 2. The sequences are compared with the classical HRPC which is 91% identical to AtPCb. The atomic structure of HRPC has been solved at 2.15 A ˚ resolution by X-ray crystallography [33]. Moreover, HRPC has been solved at 1.8 A ˚ resolution in complex with the substrate analog benzhydroxamic acid [34], and at 1.45 A ˚ resolution in the ternary complex of HRPC–cyanide–ferulic acid [35]. The structural elements of HRPC are shown in Fig. 2 in the same color as in Fig. 1 for reference. The structures of peanut peroxidase C1 [36], 67% identical to AtP49, barley grain peroxidase BP1 [37], 56% identical to AtP4, and recombinant mature AtPN [38], AtPA2 [39,40], and soybean peroxidase SBP [41], 61% identical to AtPA2 and 60% identical to AtPEa, have also been determined by X-ray crystallography. All showed the same active site structure and very similar protein folds, except for BP1 that is inactive above pH 5, and at pH 5.5, 7.5 and 8.5 has a distorted loop of 21 residues [37]. This appears to be a special feature of BP1. Active site residues of the plant peroxidase superfamily [4], shown in red in Fig. 1, include the catalytic distal Arg38, and His42 hydrogen-bonded to Asn70. In addition, the carbonyl of Pro139 accepts a hydrogen bond from reducing substrates and thereby becomes a determinant of peroxidase substrate specificity [39,40]. At the proximal site of the heme, His170 is coordinated to heme Fe 3+ and hydrogen bonded to Asp247 [42]. Many active site mutants have been designed for HRPC with the purpose of studying the function of the individual side chains (reviewed in [10,43]). Proximal His and Asp are both invariant in Fig. 1. At the distal site, the most significant substitutions occur in the 74% identical AtP50 and At5g24070 proteins, where Phe41- His42 is replaced by Tyr-Ser. The substitution of distal histidine will result in a different reaction mechanism. The change of Asn70, found in seven peroxidases, can cause a significant change in the enzyme kinetics [43]. Two stabilizing Ca 2+ ions are present in the structures of all active class III peroxidases presently known. Figure 1 shows the predicted side chain ligands in blue, and demonstrates that they are very well conserved. Main chain carbonyl oxygen and a water molecule hydrogen-bonded to the invariant Glu64 contribute other ligands. Each Ca 2+ Table 1. Annotation of the class III peroxidase gene family in Arabidopsis. Peroxidases are listed in the same similarity order as in Fig. 1, and referred to by gene accession number at MIPS, AtP name and cDNA accession number at GenBank. Underlined cDNAs were sequenced in this work; accession numbers from Ceres, Inc. are in parentheses. Positions of introns (1, 2, 3 and atypical n) and phases were predicted using the server at the Technical University of Denmark (http://www.cbs.dtu.dk/services/) and confirmed with available cDNA sequences. NETSTART and SIGNALP at this server were used for predicting start methionine residues and N-terminal signal peptides. 5¢-UTR sequences were annotated with known cDNAs and by using the NNPP program at University of California, Berkeley (http://www.fruitfly.org/seq_tools/promoter.html). The length and adenosine contentof5¢-UTRs are given from observed and predicted (o/p) data. Predicted protein length is from the most likely start methionine. Score corresponds to the maximum cleavage site probability predicted with the hidden Markov model. Underlined numbers indicate alternative predictions. pI values were calculated from the putative mature proteins truncated to HRPC residues 1–305. Peroxidase nomenclature Introns 5¢-UTR Protein Signal peptide pI Gene no. MIPS Name cDNA acc. no. Name Phase Length (o/p) A% (o/p) Start Met score Length (aa) Length Score At3g49120 AtPCb X71794 123 001 50/54 28/26 0.481 353 30 0.722 8.8 At3g49110 AtPCa AY049304 123 001 49/53 29/28 0.468 354 31 0.798 8.4 At3g32980 AtP16 X98777 123 001 44/48 39/35 0.478 352 29 0.727 7.7 At4g08770 AtP38 AF452387 123 001 11/51 55/49 0.682 346 22 0.899 8.1 At4g08780 123 001 –/52 –/46 0.534 346 22 0.900 8.1 At2g38380 AtPEa AF452388 123 001 59/62 36/34 0.830 349 29 0.629 6.0 At2g38390 AtP34 AF452385 123 001 45/49 33/35 0.844 349 29 0.655 8.7 At5g06730 AtP29 Y11794 123 001 57/66 46/44 0.692 358 31 0.581 4.8 At5g06720 AtPA2 X99952 123 001 48/75 42/39 0.757 335 30 0.333 4.8 At5g19880 AtP42 (100990) 123 001 64/64 34/34 0.760 329 23 0.736 5.0 At5g19890 AtPN X98453 123 001 67/69 45/45 0.647 321 21 0.978 6.4 At5g58390 AtP44 (124846) 12- 00- 81/83 43/42 0.293 316 19 0.957 9.9 At5g58400 12- 00- –/63 –/56 0.516 325 28 0.732 9.6 At5g05340 AtP49 AY065270 123 001 56/59 38/36 0.817 324 21 0.525 8.9 At1g14540 AtP46 AI996783 a 123 001 –/112 –/40 0.494 315 19 0.471 7.7 At1g14550 123 001 –/115 –/45 0.616 315 24 0.829 8.7 At5g66390 AtP6 X98774 123 001 40/66 50/47 0.575 336 23 0.763 8.6 At3g50990 123 001 –/52 –/42 0.457 336 21 0.764 4.7 At4g36430 AtP31 AF452384 123 001 49/52 27/27 0.608 331 22 0.982 8.8 Ó FEBS 2002 73 peroxidases from Arabidopsis (Eur. J. Biochem. 269) 6071 Table 1. (Continued). Peroxidase nomenclature Introns 5¢-UTR Protein Signal peptide pI Gene no. MIPS Name cDNA acc. no. Name Phase Length (o/p) A% (o/p) Start Met score Length (aa) Length Score At2g18140 123 001 –/97 –/29 0.865 337 22 0.473 5.8 At2g18150 AtP36 AF451952 123 001 66/69 27/26 0.901 338 22 0.613 5.8 At1g44970 AtP18 X98804 123 001 25/25 52/52 0.894 346 23 0.691 7.0 At2g35380 AtP28 Y11793 a 12- 00- –/36 –/47 0.542 336 24 0.736 5.3 At2g22420 AtP25 Y11790 12- 00- 70/92 49/48 0.739 329 20 0.572 5.0 At1g49570 AtP5 X98809 123 001 33/54 33/35 0.706 344 21 0.464 5.6 At1g68850 AtP23 Y11789 123 001 56/74 43/39 0.613 336 20 0.929 5.1 At4g16270? 123 101 –/66 –/42 0.618 21 0.737 At1g71695 AtP4 X98773 12- 00- 54/54 65/65 0.779 358 31 0.515 8.4 At5g42180 AtP17 X99096 123 001 70/74 43/43 0.864 317 22 0.869 9.2 At5g51890 AtP27 Y11792 12- 00- 66/66 44/44 0.886 322 24 0.803 9.4 At4g33420 AtP32 AF451951 123 001 57/57 42/42 0.627 314 25 0.385 5.8 At4g33870? 1n3n 0202 –/79 –/42 0.452 24 0.207 At5g64100 AtP3 X98808 ) 23 ) 01 61/64 51/52 0.860 331 23 0.870 9.1 At5g64110 AtP45 AY065173 ) 23 ) 01 84/89 52/53 0.745 330 24 0.588 6.1 At5g64120 AtP15 X99097 ) 23 ) 01 56/61 45/43 0.740 328 23 0.618 8.2 At5g39580 AtP24 Y11788 ) 23 ) 01 52/83 50/47 0.920 319 22 0.755 8.7 At2g41480 123 001 –/39 –/49 0.415 328 26 0.233 6.6 At1g77100 123 001 –/26 –/27 0.249 319 22 0.990 5.0 At4g25980 1 – 0 – –/63 –/21 0.479 326 24 0.657 5.4 At5g17820 AtP13 X98776 1–n 0–2 61/66 49/47 0.811 313 22 0.952 10 At3g03670 AtP39 (41446) 1–n 0–2 28/67 46/42 0.418 321 21 0.976 4.9 At1g34510 1 – 0 – –/81 –/47 0.850 310 20 0.980 9.3 At4g26010 AtP35 AF452386 1 – 0 – 58/61 40/38 0.756 319 20 0.546 10 At5g22410 AtP14 X98803 123 001 22/38 55/47 0.778 331 26 0.645 7.0 At2g43480 AtP50 AY078928 123 001 13/60 15/25 0.817 335 25 0.542 8.7 At5g24070 123 001 –/161 –/34 0.772 340 25 0.638 7.1 At3g21770 AtP7 X98854 123 001 78/79 41/41 0.677 326 24 0.593 9.7 At1g05260 AtPRC U97684 123 001 59/59 51/51 0.767 326 24 0.771 8.8 At4g11290 AtP19 X98805 123 001 30/44 47/43 0.700 326 23 0.692 6.6 At1g05240\ AtP11 X98802 123 001 45/68 40/34 0.835 325 21 0.796 9.3 At1g05250/ At3g01190 AtP12 X98775 123 001 59/59 54/54 0.706 321 23 0.665 9.1 At5g15180 AtP33 AY072172 123 001 42/42 43/43 0.480 329 31 0.566 8.7 At2g39040 AtP47 AV554730 123 001 –/57 –/53 0.514 350 27 0.269 8.1 At4g37520 AtP9 X98314 123 001 42/44 40/41 0.622 329 25 0.938 9.0 At4g37530 AtP37 AF469928 123 001 34/37 38/38 0.762 329 25 0.947 8.4 At5g67400 AtP10 X98928 123 001 40/64 43/41 0.823 329 25 0.846 9.4 At3g49960 AtP21 X98807 123 001 48/78 23/24 0.793 329 25 0.732 9.4 At4g30170 AtP8 X98855 123 001 81/84 38/38 0.650 325 25 0.804 9.4 At2g18980 AtP22 Y08781 123 001 6/23 33/39 0.193 323 23 0.921 9.6 At5g14130 AtP20 X98806 12- 00- 39/97 59/42 0.726 330 30 0.828 4.9 At5g40150 AtP26 Y11791 a – – –/151 –/21 0.881 328 27 0.909 8.6 At3g28200 AtP41 AY034973 – – 12/15 25/20 0.719 316 19 0.641 9.2 At5g47000 AtP43 AY093131 – – 167/170 26/26 0.782 331 25 0.824 6.8 At4g17690 – – –/99 –/32 0.821 326 20 0.987 8.4 At1g24110 – – –/362 –/39 0.160 326 20 0.453 6.1 At2g34060 AtP51 AY080602 a 12- 00- –/18 –/39 0.355 346 31 0.259 9.1 At3g17070 AtP40 (155041) 1–3 0–1 53/130 42/32 0.568 339 28 0.735 4.8 At1g30870 AtP30 AA067592 1 – 0 – 50/57 54/54 0.729 349 22 0.526 7.7 At2g24800 123 001 –/186 –/30 0.494 329 29 0.793 5.0 At4g31760 AtP48 AI999763 a 123 001 –/365 –/32 0.658 326 26 0.332 4.6 At4g21960 AtP1 X98189 123 001 70/76 39/38 0.905 330 27 0.387 8.1 At2g37130 AtP2 X98190 n123 2001 54/56 54/54 0.362 327 28 0.436 6.8 At1g34330 pseudogene At3g42570 pseudogene a Nonfull-length cDNA. 6072 K. G. Welinder et al.(Eur. J. Biochem. 269) Ó FEBS 2002 [...]... ortholog of HRPC2 (91% amino acid identity) Shinmyo et al [64] have studied the promoter activity and wound-induction of HRPC and -E types, and of AtPCa and AtPEa Most remarkably, C2, and only C2, responded strongly to wounding The AtP38 gene might respond similarly The Arabidopsis genome encodes two 84% identical anionic A-type peroxidases (Supplementary material, Table S3) AtPA2 is followed in tandem by... majority of the peroxidases carry one or two putative glycans Seven appear to be nonglycosylated Therefore, the high number of glycans found in HRP C, E and A types is unusual among class III peroxidases Since glycans are large, those close to substrate-binding residues (near Pro139) are likely to affect substrate access and reaction dynamics, due to a dampening of backbone motion [40] Half of the... of the proximal domains of the AtP50 and At5g24070 proteins have only one negatively charged aspartate, and might bind a monovalent cation similar to some class I ascorbate peroxidases [5] The presence of four disulfide bridges linking HRPC cysteine residues, 11–91, 44–49, 97–301, and 177–209 are conserved in class III peroxidases only, and highlighted in dark yellow color in Fig 1 The last Cys301 of. .. Vind, J & Dalbøge, H (1993b) The sequence of Coprinus peroxidase gene ctp1 In Plant Peroxidases Biochemistry and Physiology (Welinder, K.G., Rasmussen, S.K., Penel, C & Greppin, H., eds), pp 239–242 University of Geneva, Switzerland Reddy, C.A (1993) An overview of recent advances on the physiology and molecular biology of lignin peroxidases of Phanerochaete chrysosporium J Biotechnol 30, 91–107 Penel,... for peroxidases from other Brassicaceae, whereas Solanaceae, including potato, tomato and tobacco, Fabaceae, including soybean and peanut, and Poaceae, including barley and rice, have additional groups and subgroups of paralogous peroxidase genes, as illustrated by the few examples mentioned throughout this paper A phylogenetic analysis of class III peroxidases will appear in a separate paper 73 peroxidases. .. analysis of cDNA in Arabidopsis thaliana, generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries DNA Res 7, 175–180 The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana Nature 408, 796–815 Tognolli, M., Penel, C., Greppin, H & Simon, P (2002) Analysis and expression of the class III peroxidase... Shannon, L.M., Kay, E & Lew, J .Y (1966) Peroxidase isoenzymes from horseradish root J Biol Chem 241, 2166– 2172 64 Shinmyo, A., Fujiyama, K., Kawaoka, A & Intapruk, C (1993) Structure and expression of peroxidase isozyme genes in horseradish and Arabidopsis In Plant Peroxidases, Biochemistry and Physiology (Welinder, K.G., Rasmussen, S.K., Penel, C & 73 peroxidases from Arabidopsis (Eur J Biochem 269)... independent estimate of gene transcription that is also not biased by gene similarity On average, 0.85% of all Arabidopsis ESTs presently known encode a class III peroxidase Table 4 indicates that the level of transcription of peroxidase genes varies tremendously, from zero to 181 ESTs Twenty-nine of the 49 different AtP ESTs have been seen > 5 times We consider libraries > 10 000 and EST counts > 5... (1997) From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase Biochem J 326, 305–310 Baunsgaard, L., Dalbøge, H., Houen, G., Rasmussen, E.M & Welinder, K.G (1993a) Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase A new family of fungal peroxidases. .. attachment of the N- and C-terminal domains during protein folding Several other residues are either invariant or highly conserved (Fig 1) in class III peroxidases, Leu2, Tyr7, Pro12, Ile17, Phe41, Gly48, Glu64, Gly76, Phe77, Lys84, Ó FEBS 2002 Glu88, Pro92, Val95, Ala98, Gly114, Pro115, Asp125, Phe152, Asp161, Leu166, Gly168, Gly173, Arg183, Gly242, Leu250, Phe273, Phe277, Met281, Gly295, Arg298 and Asn305 . Differential activity and structure of highly similar peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase. University of Geneva, Switzer- land. 8. Reddy, C.A. (1993) An overview of recent advances on the physiology and molecular biology of lignin peroxidases of Phan- erochaete