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Karen G Welinder

Publications and source records attributed to Karen G Welinder.

12 recordsLinked to original sources
PubMed2006

Molecular properties and activities of tuber proteins from starch potato cv. Kuras.

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.

Electrophoresis, Polyacrylamide Gel↗
PubMed2006

Roles of distal arginine in activity and stability of Coprinus cinereus peroxidase elucidated by kinetic and NMR analysis of the Arg51Gln, -Asn, -Leu, and -Lys mutants.

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.

Amino Acid Substitution↗
PubMed2006

Patatins, Kunitz protease inhibitors and other major proteins in tuber of potato cv. Kuras.

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.

Amino Acid Sequence↗
PubMed2005

Global transcript profiling of potato tuber using LongSAGE.

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.

Journal Article↗
PubMed2004

Use of performic acid oxidation to expand the mass distribution of tryptic peptides.

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.

Amino Acids↗
PubMed2004

Covalent structure of soybean seed coat peroxidase.

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.

Amino Acid Sequence↗
PubMed2004

Database-independent, database-dependent, and extended interpretation of peptide mass spectra in VEMS V2.0.

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.

Algorithms↗
PubMed2003

Relationships of ligand binding, redox properties, and protonation in Coprinus cinereus peroxidase.

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.

Binding Sites↗
PubMed2003

The peroxidase gene family in plants: a phylogenetic overview.

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.

Arabidopsis↗
PubMed2002

Fine-tuning of the binding and dissociation of CO by the amino acids of the heme pocket of Coprinus cinereus peroxidase.

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.

Amino Acid Substitution↗
PubMed2002

Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana.

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.

Amino Acid Sequence↗