scholarly journals From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase

1997 ◽  
Vol 326 (2) ◽  
pp. 305-310 ◽  
Author(s):  
Hans M. JESPERSEN ◽  
Inger V. H. KJÆRSGÅRD ◽  
Lars ØSTERGAARD ◽  
Karen G. WELINDER
Keyword(s):  
Ascorbate Peroxidase ◽  
Sequence Data ◽  
Duplication Event ◽  
Monovalent Cation ◽  
Early Gene ◽  
Active Site Residues ◽  
Cation Site ◽  
Ascorbate Peroxidases ◽  
Complete Sequencing ◽  
Membrane Spanning

Ascorbate peroxidases are haem proteins that efficiently scavenge H2O2 in the cytosol and chloroplasts of plants. Database analyses retrieved 52 expressed sequence tags coding for Arabidopsis thalianaascorbate peroxidases. Complete sequencing of non-redundant clones revealed three novel types in addition to the two cytosol types described previously in Arabidopsis. Analysis of sequence data available for all plant ascorbate peroxidases resulted in the following classification: two types of cytosol soluble ascorbate peroxidase designated cs1 and cs2; three types of cytosol membrane-bound ascorbate peroxidase, namely cm1, bound to microbodies via a C-terminal membrane-spanning segment, and cm2 and cm3, both of unknown location; two types of chloroplast ascorbate peroxidase with N-terminal transit sequences, the stromal ascorbate peroxidase (chs), and the thylakoid-bound ascorbate peroxidase showing a C-terminal transmembrane segment and designated cht. Further comparison of the patterns of conserved residues and the crystal structure of pea ascorbate peroxidase showed that active site residues are conserved, and three peptide segments implicated in interaction with reducing substrate are similar, excepting cm2 and cm3 types. A change of Phe-175 in cytosol types to Trp-175 in chloroplast types might explain the greater ascorbate specificity of chloroplast compared with cytosol ascorbate peroxidases. Residues involved in homodimeric subunit interaction are conserved only in cs1, cs2 and cm1 types. The proximal cation (K+)-binding site observed in pea ascorbate peroxidase seems to be conserved. In addition, cm1, cm2, cm3, chs and cht ascorbate peroxidases contain Asp-43, Asn-57 and Ser-59, indicative of a distal monovalent cation site. The data support the hypothesis that present-day peroxidases evolved by an early gene duplication event.

scholarly journals Ancient and modern duplication events and the evolution of stearoyl-CoA desaturases in teleost fishes

2008 ◽  
Vol 35 (1) ◽  
pp. 18-29 ◽  
Author(s):  
Helen Evans ◽  
Tony De Tomaso ◽  
Mike Quail ◽  
Jane Rogers ◽  
Andrew Y. Gracey ◽  
...  
Keyword(s):  
Common Carp ◽  
Fatty Acid Biosynthesis ◽  
Expressed Sequence Tag ◽  
Sequence Data ◽  
Phylogenetic Analyses ◽  
Hormonal Treatment ◽  
Duplication Event ◽  
Teleost Fishes ◽  
Model Species ◽  
Duplication Events

Stearoyl-CoA desaturases (SCDs) are key enzymes of fatty acid biosynthesis whose regulation underpins responses to dietary, thermal, and hormonal treatment. Although two isoforms are known to exist in the common carp and human and four in mouse, there is no coherent view on how this gene family evolved to generate functionally diverse members. Here we identify numerous new SCD homologs in teleost fishes, using sequence data from expressed sequence tag (EST) and cDNA collections and genomic model species. Phylogenetic analyses of the deduced coding sequences produced only partially resolved molecular trees. The multiple SCD isoforms were, however, consistent with having arisen by an ancient gene duplication event in teleost fishes together with a more recent duplication in the tetraploid carp and possibly also salmonid lineages. Critical support for this interpretation comes from comparison across all vertebrate groups of the gene order in the genomic environments of the SCD isoforms. Using syntenically aligned chromosomal fragments from large-insert clones of common carp and grass carp together with those from genomically sequenced model species, we show that the ancient and modern SCD duplication events in the carp lineage were each associated with large chromosomal segment duplications, both possibly linked to whole genome duplications. By contrast, the four mouse isoforms likely arose by tandem duplications. Each duplication in the carp lineage gave rise to differentially expressed SCD isoforms, either induced by cold or diet as previously shown for the recent duplicated carp isoforms or tissue specific as demonstrated here for the ancient duplicate zebrafish isoforms.

scholarly journals Lipid hydroperoxides permit deformation-dependent leak of monovalent cation from erythrocytes

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2757-2763
Author(s):  
T Sugihara ◽  
W Rawicz ◽  
EA Evans ◽  
RP Hebbel
Keyword(s):  
Lipid Hydroperoxide ◽  
Monovalent Cation ◽  
Permeability Barrier ◽  
Lipid Hydroperoxides ◽  
Hydrophobic Membrane ◽  
Cation Homeostasis ◽  
Parallel Control ◽  
Membrane Spanning ◽  
Leak Pathway ◽  
Membrane Defect

Subtle peroxidative perturbation of normal red blood cells (RBC) using t-butylhydroperoxide creates a leak pathway for monovalent cations that is reversibly activated by cell deformation. To determine what factor promotes expression of this unique membrane defect, we have dissected “peroxidation” into components that can be evaluated separately by comparing K leak from suitably modified RBC during elliptical deformation and parallel control incubation. Selective introduction of phospholipid hydroperoxides into normal RBC membranes successfully induces a deformation-dependent leak pathway having the same phenomenology as that previously documented for cells treated with t- butylhydroperoxide itself (fully recoverable; calcium-independent; inhibited at lower pH; K efflux balanced by Na influx). This leak pathway occurs in the absence of detectable secondary peroxidative change and appears to reflect a direct influence of lipid hydroperoxide. Using micropipette examination of vesicular bilayers reconstituted from RBC lipid extracts, we find that lipid from peroxidized RBC exhibits only a slight tendency to be less cohesive than normal lipid, apparently precluding isolated lipid properties as an explanation for altered permeability barrier function. However, addition of a hydrophobic membrane-spanning peptide to these same lipids significantly diminishes bilayer cohesion, an effect that is exacerbated further by the presence of peroxidized lipid. These observations suggest that lipid hydroperoxide is a necessary, but perhaps not sufficient, factor for induction of this unique leak pathway. Our results may be relevant to the abnormal cation homeostasis of sickle RBC in which deformation of an oxidatively perturbed membrane occurs during the sickling phenomenon.

scholarly journals Analysis of the Topology and Active-Site Residues of WbbF, a Putative O-Polysaccharide Synthase from Salmonella enterica Serovar Borreze

2019 ◽  
Vol 202 (5) ◽  
Author(s):  
Samantha S. Wear ◽  
Brittany A. Hunt ◽  
Bradley R. Clarke ◽  
Chris Whitfield
Keyword(s):  
Salmonella Enterica ◽  
Lipid A ◽  
Critical Role ◽  
Cellulose Synthase ◽  
Site Directed Mutagenesis ◽  
Chain Extension ◽  
Active Site Residues ◽  
Content Type ◽  
Membrane Spanning ◽  
And Function

ABSTRACT Bacterial lipopolysaccharides are major components and contributors to the integrity of Gram-negative outer membranes. The more conserved lipid A-core part of this complex glycolipid is synthesized separately from the hypervariable O-antigenic polysaccharide (OPS) part, and they are joined in the periplasm prior to translocation to the outer membrane. Three different biosynthesis strategies are recognized for OPS biosynthesis, and one, the synthase-dependent pathway, is currently confined to a single example: the O:54 antigen from Salmonella enterica serovar Borreze. Synthases are complex enzymes that have the capacity to both polymerize and export bacterial polysaccharides. Although synthases like cellulose synthase are widespread, they typically polymerize a glycan without employing a lipid-linked intermediate, unlike the O:54 synthase (WbbF), which produces an undecaprenol diphosphate-linked product. This raises questions about the overall similarity between WbbF and conventional synthases. In this study, we examine the topology of WbbF, revealing four membrane-spanning helices, compared to the eight in cellulose synthase. Molecular modeling of the glycosyltransferase domain of WbbF indicates a similar architecture, and site-directed mutagenesis confirmed that residues important for catalysis and processivity in cellulose synthase are conserved in WbbF and required for its activity. These findings indicate that the glycosyltransferase mechanism of WbbF and classic synthases are likely conserved despite the use of a lipid acceptor for chain extension by WbbF. IMPORTANCE Glycosyltransferases play a critical role in the synthesis of a wide variety of bacterial polysaccharides. These include O-antigenic polysaccharides, which form the distal component of lipopolysaccharides and provide a protective barrier important for survival and host-pathogen interactions. Synthases are a subset of glycosyltransferases capable of coupled synthesis and export of glycans. Currently, the O:54 antigen of Salmonella enterica serovar Borreze involves the only example of an O-polysaccharide synthase, and its generation of a lipid-linked product differentiates it from classical synthases. Here, we explore features conserved in the O:54 enzyme and classical synthases to shed light on the structure and function of the unusual O:54 enzyme.

scholarly journals Lipid hydroperoxides permit deformation-dependent leak of monovalent cation from erythrocytes

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2757-2763 ◽  
Author(s):  
T Sugihara ◽  
W Rawicz ◽  
EA Evans ◽  
RP Hebbel
Keyword(s):  
Lipid Hydroperoxide ◽  
Monovalent Cation ◽  
Permeability Barrier ◽  
Lipid Hydroperoxides ◽  
Hydrophobic Membrane ◽  
Cation Homeostasis ◽  
Parallel Control ◽  
Membrane Spanning ◽  
Leak Pathway ◽  
Membrane Defect

Abstract Subtle peroxidative perturbation of normal red blood cells (RBC) using t-butylhydroperoxide creates a leak pathway for monovalent cations that is reversibly activated by cell deformation. To determine what factor promotes expression of this unique membrane defect, we have dissected “peroxidation” into components that can be evaluated separately by comparing K leak from suitably modified RBC during elliptical deformation and parallel control incubation. Selective introduction of phospholipid hydroperoxides into normal RBC membranes successfully induces a deformation-dependent leak pathway having the same phenomenology as that previously documented for cells treated with t- butylhydroperoxide itself (fully recoverable; calcium-independent; inhibited at lower pH; K efflux balanced by Na influx). This leak pathway occurs in the absence of detectable secondary peroxidative change and appears to reflect a direct influence of lipid hydroperoxide. Using micropipette examination of vesicular bilayers reconstituted from RBC lipid extracts, we find that lipid from peroxidized RBC exhibits only a slight tendency to be less cohesive than normal lipid, apparently precluding isolated lipid properties as an explanation for altered permeability barrier function. However, addition of a hydrophobic membrane-spanning peptide to these same lipids significantly diminishes bilayer cohesion, an effect that is exacerbated further by the presence of peroxidized lipid. These observations suggest that lipid hydroperoxide is a necessary, but perhaps not sufficient, factor for induction of this unique leak pathway. Our results may be relevant to the abnormal cation homeostasis of sickle RBC in which deformation of an oxidatively perturbed membrane occurs during the sickling phenomenon.

scholarly journals Exploiting protein structure data to explore the evolution of protein function and biological complexity

2006 ◽  
Vol 361 (1467) ◽  
pp. 425-440 ◽  
Author(s):  
Russell L Marsden ◽  
Juan A.G Ranea ◽  
Antonio Sillero ◽  
Oliver Redfern ◽  
Corin Yeats ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Active Sites ◽  
Sequence Data ◽  
Protein Biosynthesis ◽  
X Ray Crystallography ◽  
Metabolism Regulation ◽  
Domain Structures ◽  
Universal Domain ◽  
Complete Sequencing

New directions in biology are being driven by the complete sequencing of genomes, which has given us the protein repertoires of diverse organisms from all kingdoms of life. In tandem with this accumulation of sequence data, worldwide structural genomics initiatives, advanced by the development of improved technologies in X-ray crystallography and NMR, are expanding our knowledge of structural families and increasing our fold libraries. Methods for detecting remote sequence similarities have also been made more sensitive and this means that we can map domains from these structural families onto genome sequences to understand how these families are distributed throughout the genomes and reveal how they might influence the functional repertoires and biological complexities of the organisms. We have used robust protocols to assign sequences from completed genomes to domain structures in the CATH database, allowing up to 60% of domain sequences in these genomes, depending on the organism, to be assigned to a domain family of known structure. Analysis of the distribution of these families throughout bacterial genomes identified more than 300 universal families, some of which had expanded significantly in proportion to genome size. These highly expanded families are primarily involved in metabolism and regulation and appear to make major contributions to the functional repertoire and complexity of bacterial organisms. When comparisons are made across all kingdoms of life, we find a smaller set of universal domain families (approx. 140), of which families involved in protein biosynthesis are the largest conserved component. Analysis of the behaviour of other families reveals that some (e.g. those involved in metabolism, regulation) have remained highly innovative during evolution, making it harder to trace their evolutionary ancestry. Structural analyses of metabolic families provide some insights into the mechanisms of functional innovation, which include changes in domain partnerships and significant structural embellishments leading to modulation of active sites and protein interactions.

scholarly journals New Genomics Resources for Strawberry

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1146E-1147
Author(s):  
Thomas M. Davis ◽  
Kevin M. Folta ◽  
M. M. Shields ◽  
Robin L. Brese ◽  
Laura M. R. DiMeglio ◽  
...  
Keyword(s):  
Sequence Data ◽  
Group Structure ◽  
Nuclear Genome ◽  
Single Copy ◽  
Structural Genomic ◽  
Genomic Tools ◽  
Substantial Progress ◽  
Species Comparisons ◽  
Complete Sequencing ◽  
Rosaceae Family

The past year has brought substantial progress in the development of functional and structural genomic tools for strawberry. Sequencing of cDNA library clones from the cultivated strawberry Fragaria × ananassa and the diploid model species Fragaria vesca has provided more than 3000 new EST sequences. We have also constructed a large (∼40 kb) insert genomic (fosmid) library from F. vesca. About 33,000 fosmid clones have been picked and spotted onto hybridization filters. Filters have been successfully probed with three single copy gene probes, one gene family probe, and chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) probe sets. The combined cpDNA and mtDNA clone content of the library is about 11%. After correction for organelle insert content, the nuclear genome coverage of the library is about 6×. Complete sequencing of two fosmid clones identified 12 putative protein-encoding genes, four of which were organized in colinearity with the corresponding chromosomal region of Arabidopsis thaliana. We will sequence an additional 50 fosmid clones, and use the resulting sequence data as the basis for developing a novel marker technology, to be described. These genomic tools will provide a basis for connecting specific genes to specific traits in the octoploid, cultivated strawberry, paving the way for implementation of gene-based, marker assisted selection as a tool for strawberry breeders. Opportunity for cross-species comparisons of gene sequence and composition, as well as genome organization and linkage group structure, between Fragaria and other members of the economically important Rosaceae family has been significantly enhanced, thus expanding the relevance of the project results to peach, cherry, apple, rose, brambles, and many other Rosaceous species.

scholarly journals Cloning, in vitro mitochondrial import and membrane assembly of the 17.8 kDa subunit of complex I from Neurospora crassa

1993 ◽  
Vol 293 (2) ◽  
pp. 501-506 ◽  
Author(s):  
J E Azevedo ◽  
J Abrolat-Scharff ◽  
C Eckerskorn ◽  
S Werner
Keyword(s):  
Neurospora Crassa ◽  
Complex I ◽  
Sequence Data ◽  
Membrane Topology ◽  
Intermembrane Space ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Intermembrane Space ◽  
Processing Peptidase ◽  
Membrane Spanning

We have cloned and sequenced a cDNA encoding a 17.8 kDa subunit of the hydrophobic fragment of complex I from Neurospora crassa. The deduced primary structure of this subunit was partially confirmed by automated Edman degradation of the isolated polypeptide. The sequence data obtained indicate that the 17.8 kDa subunit is made as an extended precursor of 20.8 kDa. Resistance of the polypeptide to alkaline extraction from mitochondrial membranes and the existence of a putative membrane-spanning domain suggests that the 17.8 kDa subunit is an intrinsic (bitopic) membrane protein. The in vitro synthesized precursor of the 17.8 kDa subunit can be efficiently imported into isolated mitochondria, where it is cleaved to the mature species by the metal-dependent matrix-processing peptidase. The in vitro imported mature subunit is found mainly exposed to the mitochondrial intermembrane space. However, a significant fraction of the imported polypeptide acquires the same membrane topology as the endogenous subunit, indicating that correct assembly in the mitochondrial inner membrane did occur.

scholarly journals Tryptophan Synthase: Structure and Function of the Monovalent Cation Site

Biochemistry ◽  
2009 ◽  
Vol 48 (46) ◽  
pp. 10997-11010 ◽  
Author(s):  
Adam T. Dierkers ◽  
Dimitri Niks ◽  
Ilme Schlichting ◽  
Michael F. Dunn
Keyword(s):  
Monovalent Cation ◽  
Structure And Function ◽  
Tryptophan Synthase ◽  
Cation Site ◽  
And Function

scholarly journals Cold priming memory reduces plant pathogen susceptibility based on a functional plastid peroxidase system

2020 ◽  
Author(s):  
Thomas Griebel ◽  
Alina Ebert ◽  
Hoang Hung Nguyen ◽  
Margarete Baier
Keyword(s):  
Pseudomonas Syringae ◽  
Ascorbate Peroxidase ◽  
Cold Exposure ◽  
Peroxidase Activity ◽  
Plant Pathogen ◽  
Gene Activation ◽  
Transcriptional Response ◽  
Immune Gene ◽  
Memory Element ◽  
Ascorbate Peroxidases

ABSTRACTChloroplasts, as recently shown, serve as cold priming hubs in modulating the transcriptional response of Arabidopsis thaliana to a second cold stimulus after a stress-free interphase of several days. Here, we studied if such a single 24 h cold stress at 4 °C also alters the susceptibility of Arabidopsis to virulent Pseudomonas syringae pv. tomato DC3000. Our data show that cold priming did not only increase resistance of Arabidopsis to a subsequent infection immediately, but also after a memory phase of 5 days, during which thylakoid ascorbate peroxidases accumulate. Contrasting to susceptibility, the prior cold exposure did not alter resistance against avirulent and effector-triggered immunity-inducing Pseudomonas syringae strains. The effect of cold priming on the plant pathogen susceptibility was independent of the central nucleo-cytoplasmic immune regulator EDS1 (Enhanced Disease Susceptibility 1) and uncoupled from classical immune gene activation. The priming benefits against pathogens required thylakoid and stromal ascorbate peroxidase activity. Combinatorial priming of Arabidopsis pathogen susceptibility by metastable regulation of stromal ascorbate peroxidase activity and post-cold expression of thylakoid ascorbate peroxidase guarantees immediate protection without latency time and prolonged protection by the memory element that regulates future cold responses.One-sentence summary24 hour cold exposure reduces plant susceptibility against virulent pathogens dependent on chloroplast ascorbate peroxidases.

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