RESUMO
Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic analyses indicated that PAPhys possess four conserved domains unique to the PAPhys. In barley and wheat, the PAPhy genes can be grouped as PAPhy_a or PAPhy_b isogenes (barley, HvPAPhy_a, HvPAPhy_b1, and HvPAPhy_b2; wheat, TaPAPhy_a1, TaPAPhy_a2, TaPAPhy_b1, and TaPAPhy_b2). In rice and maize, only the b type (OsPAPhy_b and ZmPAPhy_b, respectively) were identified. HvPAPhy_a and HvPAPhy_b1/b2 share 86% and TaPAPhya1/a2 and TaPAPhyb1/b2 share up to 90% (TaPAPhy_a2 and TaPAPhy_b2) identical amino acid sequences. despite of this, PAPhy_a and PAPhy_b isogenes are differentially expressed during grain development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed that the PAPhy_a isogene set present in wheat/barley but not in rice/maize is the origin of high phytase activity in mature grains.
Assuntos
6-Fitase/genética , Fosfatase Ácida/genética , Glicoproteínas/genética , Plantas/enzimologia , Plantas/genética , 6-Fitase/química , 6-Fitase/metabolismo , Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Germinação , Glicoproteínas/química , Glicoproteínas/metabolismo , Hordeum/enzimologia , Hordeum/genética , Cinética , Dados de Sequência Molecular , Oryza/enzimologia , Oryza/genética , Filogenia , Sementes/citologia , Sementes/enzimologia , Sementes/ultraestrutura , Alinhamento de Sequência , Espectrometria de Massas em Tandem , Fatores de Tempo , Triticum/citologia , Triticum/enzimologia , Triticum/genética , Triticum/ultraestrutura , Zea mays/enzimologia , Zea mays/genéticaRESUMO
In this article, we present mass-spectrometry based plasma proteomics data from hibernating and active free-ranging Scandinavian brown bears (Ursus arctos). The brown bear hibernates for half the year. Despite obesity when entering the den and the prolonged period of inactivity, the bear shows no signs of the harmful effects associated with these conditions in humans. Thus, the hibernating bear is a potential translational model for addressing these complications in humans. We analyzed plasma samples from fourteen 2- to 3-year-old bears (6 males and 8 females) collected both during hibernation and the active state, and for some of the bears during two seasons, resulting in a total of 38 analyzed plasma samples. In triplicates, the plasma proteins were unfolded and reduced. To increase the chance of detecting low-molecular-weight proteins and peptides, we filtered the samples using a 50 K molecular weight cut-off filter with the aim to deplete larger abundant proteins, including albumin, and thereby increase the depth of the analysis. The proteins in the permeate were then tryptically digested, desalted, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein identification and quantification was performed with the MaxQuant software searching against an Ursus arctos horribilis protein database. Here, we provide the raw data, a list with identified proteins in the plasma samples, and the databases applied for protein identification. Based on the provided data, differentially expressed proteins in hibernation compared to active state can be identified. These proteins may be involved in the bears' adaptions to hibernation physiology and hold potential as novel therapeutic targets.
RESUMO
Paramagnetic (13)C and (15)N nuclear magnetic resonance (NMR) spectroscopy of heme-bound cyanide ((13)C(15)N) was applied to 11 cytochrome c peroxidase (CcP) and Coprinus cinereus peroxidase (CIP) mutants to investigate contributions to the push and pull effects of conserved amino acids around heme. The (13)C and (15)N NMR data for the distal His and Arg mutants indicated that distal His is the key amino acid residue creating the strong pull effect and that distal Arg assists. The mutation of distal Trp of CcP to Phe, the amino acid at this position in CIP, changed the push and pull effects so they resembled those of CIP, whereas the mutation of distal Phe of CIP to Trp changed this mutant to become CcP-like. The (13)C NMR shifts for the proximal Asp mutants clearly showed that the proximal Asp-His hydrogen bonding strengthens the push effect. However, even in the absence of a hydrogen bond, the push effect of proximal His in peroxidase is significantly stronger than in globins. Comparison of these NMR data with the compound I formation rate constants and crystal structures of these mutants showed that (1) the base catalysis of the distal His is more critical for rapid compound I formation than its acid catalysis, (2) the primary function of the distal Arg is to maintain the distal heme pocket in favor of rapid compound I formation via hydrogen bonding, and (3) the push effect is the major contributor to the differential rates of compound I formation in wild-type peroxidases.
Assuntos
Coprinus/enzimologia , Citocromo-c Peroxidase/química , Heme/química , Peroxidases/química , Aminoácidos/genética , Aminoácidos/metabolismo , Catálise , Coprinus/metabolismo , Citocromo-c Peroxidase/genética , Citocromo-c Peroxidase/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Heme/genética , Heme/metabolismo , Ligação de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Peroxidases/genética , Peroxidases/metabolismoRESUMO
Potato (Solanum tuberosum) is the fourth largest crop worldwide in yield, and cv. Kuras is the major starch potato of northern Europe. Storage starch is packed densely in tuber amyloplasts, which become starch granules. Amyloplasts of soil-grown mini-tubers and agar-grown micro-tubers of cv. Kuras were purified. The mini-tuber amyloplast preparation was enriched 10-20-fold and the micro-tuber amyloplast approximately fivefold over comparative total protein extracts. Proteins separated by SDS-PAGE were digested with trypsin, analysed by mass spectrometry and identified by mascot software searches against an in-house potato protein database and the NCBI non-redundant plant database. The differential growth conditions for mini- and micro-tubers gave rise to rather different protein profiles, but the major starch granule-bound proteins were identical for both and dominated by granule-bound starch synthase I, starch synthase II and alpha-glucan water dikinase. Soluble proteins were dominated by starch phosphorylase L-1, other large proteins of the classes 'starch and sucrose metabolism', 'pentose phosphate pathway', 'glycolysis', 'amino acid metabolism', and other proteins such as plastid chaperonins. The majority of the identified proteins had a predicted plastid transit peptide, supporting their presence in the amyloplast. However, several highly expressed proteins had no transit peptide, such as starch phosphorylase H, or had a predicted mitochondrial location. Intriguingly, all polyphenol oxidases, a family of enolases, one transketolase, sulfite reductase, deoxynucleoside kinase-like and dihydroxy-acid dehydrase had twin-arginine translocation motifs, and a homologue to dihydrolipoamide dehydrogenase had a Sec (secretory) motif; these motifs usually target thylakoid-like structures.
Assuntos
Tubérculos/metabolismo , Plastídeos/metabolismo , Proteoma/metabolismo , Solanum tuberosum/metabolismo , Espectrometria de Massas , Tubérculos/química , Plastídeos/química , Proteoma/química , Solanum tuberosum/química , Amido/metabolismo , Sacarose/metabolismoRESUMO
BACKGROUND: During gene expression analysis by Serial Analysis of Gene Expression (SAGE), duplicate ditags are routinely removed from the data analysis, because they are suspected to stem from artifacts during SAGE library construction. As a consequence, naturally occurring duplicate ditags are also removed from the analysis leading to an error of measurement. RESULTS: An algorithm was developed to analyze the differential occurrence of SAGE tags in different ditag combinations. Analysis of a pancreatic acinar cell LongSAGE library showed no sign of a general amplification bias that justified the removal of all duplicate ditags. Extending the analysis to 10 additional LongSAGE libraries showed no justification for removal of all duplicate ditags either. On the contrary, while the error introduced in original SAGE by removal of naturally occurring duplicate ditags is insignificant, it leads to an error of up to 3 fold in LongSAGE. However, the algorithm developed for the analysis of duplicate ditags was able to identify individual artifact ditags that originated from rare nucleotide variations of tags and vector contamination. CONCLUSION: The removal of all duplicate ditags was unfounded for the datasets analyzed and led to large errors. This may also be the case for other LongSAGE datasets already present in databases. Analysis of the ditag population, however, can identify artifact tags that should be removed from analysis or have their tag count adjusted.
Assuntos
Etiquetas de Sequências Expressas , Regulação da Expressão Gênica/genética , Software , Algoritmos , Sequência de Bases/genética , Etiquetas de Sequências Expressas/metabolismoRESUMO
In heme peroxidases, a distal His residue plays an essential role in the initial two electron oxidation of resting state enzyme to compound I by hydrogen peroxide. A distal Arg residue assists in this process. The contributions of the charge, H-bonding capacity, size, and mobility of this Arg residue to Coprinus cinereus peroxidase (CIP) reactivity and stability have been examined by substituting Arg51 with Gln (retains H-bond donor at N epsilon position), Asn (small size, H-bond donor and acceptor), Leu (similar to Asn, but hydrophobic), and Lys (charge and H-bond donor, but at N zeta position). UV-visible spectroscopy was used to monitor pH-linked heme changes, compound I formation and reduction, fluoride binding, and thermostability. (1)H NMR spectroscopy enabled heme pocket differences in both resting and cyanide-ligated states of the enzymes to be evaluated and compared with wild-type CIP. We found that the H-bonding capacity of distal Arg is key to fast compound I formation and ligand binding to heme, whereas charge is important for lowering the pK(a) of distal His and for the binding and stabilisation of anionic ligands at heme iron. The properties of the distal Arg residue in CIP, cytochrome c peroxidase (CCP) and horseradish peroxidase (HRP) differ significantly in their pH induced transitions and dynamics.
Assuntos
Coprinus/enzimologia , Peroxidases/química , Peroxidases/metabolismo , Substituição de Aminoácidos , Arginina/química , Domínio Catalítico , Coprinus/genética , Estabilidade Enzimática , Heme/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Peroxidases/genética , EspectrofotometriaRESUMO
The major potato tuber proteins of the Kuras cultivar, which is the dominant cultivar used in Northern Europe for industrial starch production, were analysed using 1D and 2D gel electrophoresis. The electrophoretic patterns varied significantly depending on the method of preparation and the potato variant (Solanum tuberosum). Proteins were characterized using MS and scored against potato protein databases, derived from both 'Kuras only' and 'all potato' expressed sequence tags (EST) and full-length cDNAs. Despite the existence of approximately 180 000 ESTs, the currently available potato sequence data showed a severe under-representation of genes or long transcripts encoding proteins > 50 kDa (3.5% of all) compared with the complete proteome of Arabidopsis thaliana (33% of all). We found that patatin and Kunitz protease inhibitor (KPI) variants are extraordinarily dominant in Kuras tuber and, most significantly, that their amino acid sequences are specific to Kuras. Other proteins identified include annexin, glyoxalase I, enolase and two lipoxygenases, the sequences of which are highly conserved among potato variants. Known S. tuberosum patatins cluster into three clades all represented in Kuras. S. tuberosum KPIs cluster into more diverse clades of which five were found in Kuras tuber, including a novel clade, KPI K, found to date only in Kuras. Furthermore, protein abundance was contrasted with the levels of corresponding gene transcripts found in our previous EST and LongSAGE studies of Kuras tuber.
Assuntos
Hidrolases de Éster Carboxílico/isolamento & purificação , Peptídeos/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Solanum tuberosum/química , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/farmacologia , Eletroforese em Gel Bidimensional , Etiquetas de Sequências Expressas , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Potato starch production leaves behind a huge amount of juice. This juice is rich in protein, which might be exploited for food, biotechnological, and pharmaceutical applications. In northern Europe cv. Kuras is dominant for industrial starch production, and juice protein of freshly harvested mature tubers was fractionated by Superdex 200 gel filtration. The fractions were subjected to selected activity assays (patatin, peroxidase, glyoxalases I and II, alpha-mannosidase, inhibition of trypsin, Fusarium protease, and alcalase) and protein subunit size determination by SDS-PAGE and mass spectrometry. Proteins present in SDS-PAGE bands were identified by tryptic peptide mass fingerprinting. Protein complexes such as ribosomes and proteasomes eluted with the void volume of the gel filtration. Large proteins were enzymes of starch synthesis dominated by starch phosphorylase L-1 (ca. 4% of total protein). Five identified dimeric patatin variants (25%) coeluted with four monomeric lipoxygenase variants (10%) at 97 kDa. Protease inhibitor I variants (4%) at 46 kDa (hexamer) inhibited alcalase. Fourteen Kunitz protease inhibitor variants (30%) at 19 kDa inhibited trypsin and Fusarium protease. Carboxypeptidase inhibitor variants (5%) and defensins (5%) coeluted with phenolics. The native sizes and molecular properties were determined for 43 different potato tuber proteins, several for the first time.
Assuntos
Proteínas de Plantas/química , Tubérculos/química , Solanum tuberosum/química , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Mapeamento de Peptídeos , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Amido/químicaRESUMO
Peroxidase from soybean seed coat (SBP) is very stable at high temperature, extremes of pH, and in organic solvent. At the same time, it is highly reactive towards both organic and inorganic substrates, similar to horseradish peroxidase. SBP has a wide range of potential applications, and its structure is of particular interest for engineering purposes and as a model for stable heme peroxidases. The covalent structure of SBP has been determined by Edman sequencing and MALDI-TOF MS. SBP is a highly heterogeneous glycoprotein with MS determined masses from 39 to 41 kDa. The mature protein consists of 306 residues starting with pyrrolidone carboxylic acid. Seven glycosylation sites have been observed, although some sites were only partially glycosylated. No putative plant peroxidases were orthologous to SBP. However, SBP showed greater than 70% amino acid sequence identity to peroxidases from other legumes recruited in various defense responses.
Assuntos
Glycine max/química , Peroxidase/química , Sementes/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Sementes/enzimologia , Sementes/metabolismo , Glycine max/enzimologia , Glycine max/metabolismoRESUMO
Solanum tuberosum (potato) is the fourth major crop worldwide and is used for food, feed and biotechnological applications. To fully realize the biosynthetic potential for the production of starch, protein and metabolites, we conducted an extensive quantitative profiling of the expressed genes of mature potato tuber. A total of 58,322 serial analysis of gene expression (SAGE) tags of 19 nucleotides (nt), representing 22,233 different tags, were analysed. The 695 tags seen 10 or more times were assigned a tentative function by comparison with homologous genes. The identities of 12 'known' and 12 'unknown' transcripts were confirmed by rapid amplification of cDNA ends (RACE) cloning using the 19 nt tag as a primer. The SAGE and expressed sequence tag (EST) profiles of potato tuber were compared. Transcripts for four types of protease inhibitor, a metallothionein and a lipoxygenase were more prominent than patatin isoforms.
RESUMO
Proteome data of potato (Solanum tuberosum) tuber juice and of purified potato tuber vacuoles indicated that mature patatins may perhaps lack a C-terminal propeptide. We have confirmed this by complete mass spectrometric sequencing of a number of patatin variants as well as their N-linked complex-type glycans from the starch-rich cultivar Kuras. For this cultivar full-length patatin cDNAs have also been sequenced, as the patatin locus is highly polymorphous. It is well known that patatins are located in the vacuoles of potato tubers. Furthermore, the complex glycan structures show that the path is via the Golgi apparatus. However, the vacuolar targeting signal has never been identified for this storage and defense protein, which amounts to 25-40% of tuber protein. We propose that a six-residue C-terminal propeptide, -ANKASY-COO(-) comprises this signal. The crystallographic structure of a recombinant patatin (Rydel, T. J., Williams, J. M., Krieger, E., Moshiri, F., Stallings, W. C., Brown, S. M., Pershing, J. C., Prucell, J. P., and Alibhai, M. F. (2003) Biochemistry 42, 6696-6708), which included this propeptide thus, for the first time, shows the structure of a putative ligand of the vacuolar sorting receptor and processing enzyme responsible for patatin import.
Assuntos
Hidrolases de Éster Carboxílico/química , Proteínas de Plantas/química , Tubérculos/química , Vacúolos/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X/métodos , Complexo de Golgi/metabolismo , Ligantes , Dados de Sequência Molecular , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Polissacarídeos/química , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Solanum tuberosum , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
The 73 class III peroxidase genes in Arabidopsis thaliana were used for surveying the evolutionary relationships among peroxidases in the plant kingdom. In Arabidopsis, the 73 genes were clustered in robust similarity groups. Comparison to peroxidases from other angiosperms showed that the diversity observed in Arabidopsis preceded the radiation of dicots, whereas some clusters were absent from grasses. Grasses contained some unique peroxidase clusters not seen in dicot plants. We found peroxidases in other major groups of land plants but not in algae. This might indicate that the class III peroxidase gene family appeared with the colonization of land by plants. The present survey may be used as a rational basis for further investigating the functional roles of class III peroxidases.
Assuntos
Peroxidases/genética , Filogenia , Plantas/enzimologia , Plantas/genética , Arabidopsis/genética , Evolução Molecular , Flores/fisiologia , Genes de Plantas/genética , Genoma de Planta , Peroxidases/classificação , Alinhamento de SequênciaRESUMO
Significant identification of proteins by mass fingerprinting and partial sequencing of tryptic peptides is central to proteomics. However, peptide masses cluster with distances of approximately 1 Da. Expanding these clusters will give more peptides of unique masses, thereby identifying proteins with a higher significance. The mass clusters can be expanded downward by including more oxygen atoms in the peptides. Classic performic acid oxidation modifies three residues, Cys to CysO(3), Met to MetO(2), and Trp to TrpO(2). In this study, we compare the mass distributions of tryptic peptides computed from the predicted proteomes of Bacillus subtilis, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens modified by oxidation, reduction, and reduction followed by carboxymethylation, carboxamidomethylation, or pyridylethylation. Forty to 46% of the eukaryotic tryptic peptides contain Cys, Met, or Trp. Additionally, the importance of mass accuracy of differentially modified tryptic peptides for significant protein identification by database searches was analyzed. The results show that performic acid oxidation gives markedly extended mass distributions at mass accuracies from +/-0.002 to +/-0.25 Da for the eukaryotes. The effect of the expanded mass distribution on significant protein identification was illustrated by searching simulated mass peak lists against the databases containing oxidized and reduced tryptic peptides. The specificity of formic acid oxidation was tested experimentally, and no general adverse effects were detected. Tryptic peptides provided a 100% sequence coverage of oxidized barley grain peroxidase by LC-MS, and the sequence coverages of oxidized and carboxymethylated bovine serum albumin were similar by MALDI-TOF MS analyses.
Assuntos
Formiatos/química , Fragmentos de Peptídeos/química , Aminoácidos/química , Animais , Arabidopsis/química , Bacillus subtilis/química , Bovinos , Cromatografia Líquida , Bases de Dados de Proteínas , Drosophila melanogaster/química , Humanos , Espectrometria de Massas , Oxirredução , Proteoma , Sensibilidade e Especificidade , Soroalbumina Bovina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The Virtual Expert Mass Spectrometrist (VEMS) program package was developed for flexible, automated, and manual de novo tandem mass spectrometry (MS/MS) protein sequencing, and includes accessory programs for matrix-assisted laser desorption/ionization-mass spectrometry (MS) interpretation, and generation of protein and peptide databases. VEMS V2.0 has been developed into a fast tool for combining database-independent and -dependent protein assignments in an extended analysis of MS/MS-peptide data. MS or MS/MS data can be directly recalibrated after the first search by fitting the data to the best search result using polynomial equations. The score function is an improvement of known scoring algorithms and can be adapted for any MS instrument type. In addition, VEMS offers a novel statistical model for evaluating the significance of the protein assignment. The novel features are illustrated by the analysis of the fragmentation spectra obtained by liquid chromatrography-MS/MS analysis of peptides from an anionic peroxidase enriched protein fraction from potato root tissue. The extended analysis mode resulted in the additional assignment of spectra for nine modified tryptic peptides and nine miscleaved peptides, in addition to the 45 spectra from regular tryptic peptides. Of the nine modified peptides, three were glycosylated.
Assuntos
Bases de Dados de Proteínas , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Peptídeos/análise , Análise de Sequência de Proteína/métodos , Software , Algoritmos , Sequência de Aminoácidos , Sequência de Carboidratos , Dados de Sequência Molecular , Extratos Vegetais/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Reprodutibilidade dos Testes , Solanum tuberosum/químicaRESUMO
Resonance Raman and infrared spectra and the CO dissociation rates (k(off)) were measured in Coprinus cinereus peroxidase (CIP) and several mutants in the heme binding pocket. These mutants included the Asp245Asn, Arg51Leu, Arg51Gln, Arg51Asn, Arg51Lys, Phe54Trp, and Phe54Val mutants. Binding of CO to CIP produced different CO adducts at pH 6 and 10. At pH 6, the bound CO is H-bonded to the protonated distal His55 residue, whereas at alkaline pH, the vibrational signatures and the rate of CO dissociation indicate a distal side which is more open or flexible than in other plant peroxidases. The distal Arg51 residue is important in determining the rate of dissociation in the acid form, increasing by 8-17-fold in the Arg51 mutants compared to that for the wild-type protein. Replacement of the distal Phe with Trp created a new acid form characterized by vibrational frequencies and k(off) values very similar to those of cytochrome c peroxidase.
Assuntos
Aminoácidos/química , Monóxido de Carbono/química , Coprinus/enzimologia , Heme/química , Peroxidase/química , Substituição de Aminoácidos/genética , Aminoácidos/genética , Ânions , Arginina/genética , Asparagina/genética , Ácido Aspártico/genética , Coprinus/genética , Citocromo-c Peroxidase/química , Citocromo-c Peroxidase/genética , Compostos Ferrosos/química , Cinética , Ligantes , Mutação , Peroxidase/genética , Fenilalanina/genética , Ligação Proteica/genética , Espectrofotometria Infravermelho , Análise Espectral RamanRESUMO
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) encoding 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 horseradish 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). Furthermore, genes appearing in tandem showed distinct expression 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.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Peroxidases/química , Peroxidases/genética , Transcrição Gênica , Sequência de Aminoácidos , DNA Complementar/metabolismo , Etiquetas de Sequências Expressas , Genoma de Planta , Heme/química , Peroxidase do Rábano Silvestre/metabolismo , Íntrons , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
The pH dependence of the redox potentials and kinetics for CO association and dissociation was determined between pH 3.0 and 13.0 at 25 degrees C for the wild-type Coprinus cinereus fungal peroxidase and for a site-directed mutant in which Asp245, which is H-bonded to N delta of the imidazole of the proximal His183, was substituted with Asn. The determination of these functional properties allowed this information to be merged in a self-consistent fashion and to formulate for the first time a complete scheme employing the minimum number of groups required to describe the whole proton-linked behavior of both redox and ligand binding properties. The overall pH dependence can be accounted for by four redox- and ligand-linked groups. The proximal H-bond, which is strictly conserved in all peroxidases, will still be present in the site-specific mutant, but will no longer have an ionic character, and this event will bring about an alteration of redox equilibria and CO binding kinetics, envisaging a relevant role played by this H-bond also in modulating redox properties and ligand binding equilibria.