scholarly journals Purification and Biochemical Characterization of Sucrose synthase from the Stem of Nettle (Urtica dioica L.)

2021 ◽  
Vol 22 (2) ◽  
pp. 851
Author(s):  
Lavinia Mareri ◽  
Gea Guerriero ◽  
Jean-Francois Hausman ◽  
Giampiero Cai
Keyword(s):  
Sucrose Synthase ◽  
Cell Walls ◽  
Biochemical Characterization ◽  
Post Translational Modifications ◽  
Cell Compartments ◽  
Glucose Molecule ◽  
Key Enzyme ◽  
Diphosphate Glucose ◽  
Purification Protocol

Sucrose synthase is a key enzyme in sucrose metabolism as it saves an important part of sucrose energy in the uridine-5′-diphosphate glucose (UDP-glucose) molecule. As such it is also involved in the synthesis of fundamental molecules such as callose and cellulose, the latter being present in all cell walls of plant cells and therefore also in the gelatinous cell walls of sclerenchyma cells such as bast fibers. Given the importance of these cells in plants of economic interest such as hemp, flax and nettle, in this work we have studied the occurrence of Sucrose synthase in nettle stems by analyzing its distribution between the cytosol, membranes and cell wall. We have therefore developed a purification protocol that can allow the analysis of various characteristics of the enzyme. In nettle, Sucrose synthase is encoded by different genes and each form of the enzyme could be subjected to different post-translational modifications. Therefore, by two-dimensional electrophoresis analysis, we have also traced the phosphorylation profile of Sucrose synthase isoforms in the various cell compartments. This information paves the way for further investigation of Sucrose synthase in plants such as nettle, which is both economically important, but also difficult to study.

scholarly journals Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea

2017 ◽  
Author(s):  
Dipti D. Nayak ◽  
Nilkamal Mahanta ◽  
Douglas A. Mitchell ◽  
William W. Metcalf
Keyword(s):  
Strictly Anaerobic ◽  
Methanosarcina Acetivorans ◽  
Coenzyme M ◽  
Α Subunit ◽  
Post Translational Modifications ◽  
Physiological Characterization ◽  
Key Enzyme ◽  
Catalytic Role ◽  
Methyl Coenzyme M Reductase

AbstractThe enzyme methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α subunit of this enzyme (McrA) contains several unusual post-translational modifications, including an exceptionally rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioamide-containing natural products, we hypothesized that the archaealtfuAandycaOgenes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA in a ΔycaO-tfuAmutant of the methanogenic archaeonMethanosarcina acetivoransrevealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Physiological characterization of this mutant suggested a new role for the thioglycine modification in enhancing protein stability, as opposed to playing a direct catalytic role. The universal conservation of this modification suggests that MCR arose in a thermophilic ancestor.

Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme that Catalyzes C-C Bond Formation

2020 ◽  
Author(s):  
Mengbin Chen ◽  
Chun-Ting Liu ◽  
Yi Tang
Keyword(s):  
Amino Acids ◽  
Biosynthetic Pathway ◽  
Pyridoxal Phosphate ◽  
Biochemical Characterization ◽  
Indole Alkaloid ◽  
Building Blocks ◽  
Nucleophilic Attack ◽  
Keto Esters ◽  
Key Enzyme

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze various transformations of amino acids at alpha, beta, and gamma positions. These versatile enzymes are prominently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products, and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2<i>S, </i>6<i>S</i>)-6-methyl pipecolate <b>1</b>, from the biosynthetic pathway of indole alkaloid citrinadin. The key enzyme CndF is PLP-dependent and catalyzes synthesis of (<i>S</i>)-2-amino-6-oxoheptanoate <b>3</b> that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives <b>1</b>. Biochemical characterization of CndF showed this enzyme performs gamma-elimination of <i>O</i>-acetyl L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new C<sub>gamma</sub>-C<sub>delta</sub> bond in <b>3 </b>and complete the gamma-substitution reaction. CndF displays substrate promiscuity towards different beta-keto carboxylate and esters. Using a recombinant <i>Aspergillus </i>strain expressing CndF and CndE, feeding various alkyl-beta-keto esters led to the biosynthesis of 6-substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.

Comparative biochemical characterization of peroxidases (class III) tightly bound to the maize root cell walls and modulation of the enzyme properties as a result of covalent binding

PROTOPLASMA ◽  
2014 ◽  
Vol 252 (1) ◽  
pp. 335-343 ◽  
Author(s):  
Vesna Hadži-Tašković Šukalović ◽  
Mirjana Vuletić ◽  
Ksenija Marković ◽  
Tijana Cvetić Antić ◽  
Željko Vučinić
Keyword(s):  
Cell Walls ◽  
Biochemical Characterization ◽  
Covalent Binding ◽  
Root Cell ◽  
Maize Root ◽  
Enzyme Properties ◽  
Class Iii ◽  
Root Cell Walls

Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay

2011 ◽  
Vol 438 (2) ◽  
pp. 275-282 ◽  
Author(s):  
Marián Mazáň ◽  
Enrico Ragni ◽  
Laura Popolo ◽  
Vladimír Farkaš
Keyword(s):  
Cell Walls ◽  
Biochemical Characterization ◽  
Catalytic Properties ◽  
Degree Of Polymerization ◽  
The Novel ◽  
Fluorescent Assay ◽  
Fungal Cell ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Wall Biogenesis

BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C3(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR–LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.

scholarly journals Characterization of human herpesvirus 6 variant B immediate-early 1 protein modifications by small ubiquitin-related modifiers

2004 ◽  
Vol 85 (5) ◽  
pp. 1319-1328 ◽  
Author(s):  
Annie Gravel ◽  
Valérie Dion ◽  
Nathalie Cloutier ◽  
Jean Gosselin ◽  
Louis Flamand
Keyword(s):  
Biochemical Characterization ◽  
Human Herpesvirus ◽  
Immediate Early ◽  
Human Herpesvirus 6 ◽  
Post Translational Modifications ◽  
Sumo Conjugation ◽  
Complex Interactions ◽  
Herpesvirus 6

The human herpesvirus 6 (HHV-6) immediate-early (IE) 1 protein undergoes SUMOylation events during the infectious process. In the present work, we report that Lys-802 (K-802) of IE1 from HHV-6 variant B is the only target residue capable of conjugation to SUMO-1/SMT3C/Sentrin-1, SUMO-2/SMT3A/Sentrin-3 or SUMO-3/SMT3B/Sentrin-2 as determined by transfection and in vitro SUMOylation experiments. PolySUMOylated forms of IE1 were also observed, suggesting that SUMO branching occurs at the K-802 residue. Overexpression of SUMO-1, -2 and -3 led to an overall increase in IE1 levels, irrespective of K-802. The SUMO residues could be efficiently removed by incubating SUMOylated IE1 with SENP1, a recently identified SUMO peptidase. SUMOylation-deficient mutants of IE1 co-localized with nuclear promyelocytic leukaemia protein (PML) oncogenic domains (PODs) as efficiently as WT IE1, indicating that POD targeting is independent of IE1 SUMOylation status. However, in contrast to infection, PODs did not aggregate in IE1B-transfected cells, suggesting that other viral proteins are involved in the process. Transactivation studies indicated that IE1, in combination with IE2, could efficiently transactivate diverse promoters, independent of its SUMOylation status. Overall, the results presented provide a detailed biochemical characterization of post-translational modifications of the HHV-6 IE1 protein by SUMO peptides, contributing to our understanding of the complex interactions between herpesviruses and the SUMO-conjugation pathway.

scholarly journals Characterization and biological roles of oviduct-specific, oestrogen-dependent glycoprotein

Reproduction ◽  
2002 ◽  
pp. 355-362 ◽  
Author(s):  
WC Buhi
Keyword(s):  
Embryonic Development ◽  
Biochemical Characterization ◽  
Gene Organization ◽  
Hormonal Control ◽  
Expression Cloning ◽  
Early Cleavage ◽  
Control Of Gene Expression ◽  
Post Translational Modifications ◽  
Physiological And Biochemical

During late follicular growth, oestrus, fertilization and early embryonic development, the oviduct, under specific hormonal control, produces fluid and contributes secretory macromolecules that optimize the microenvironment for gamete maturation and transport, fertilization and early cleavage-stage embryonic development. This review describes the state of knowledge concerning the physiological and biochemical characterization of the major oviduct secreted protein, the oviduct-specific, oestrogen-dependent glycoprotein. The identification, localization within the oviduct, binding and association with oocytes, embryos and spermatozoa, hormonal control of gene expression, cloning, gene organization, protein sequences and post-translational modifications of oviduct-specific, oestrogen-dependent glycoprotein are discussed. Identification of biological functions for this glycoprotein, its interactions with spermatozoa, oocytes and embryos and its potential as a paracrine regulator of fertilization and development are also discussed.

Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme that Catalyzes C-C Bond Formation

2020 ◽  
Author(s):  
Mengbin Chen ◽  
Chun-Ting Liu ◽  
Yi Tang
Keyword(s):  
Amino Acids ◽  
Biosynthetic Pathway ◽  
Pyridoxal Phosphate ◽  
Biochemical Characterization ◽  
Indole Alkaloid ◽  
Building Blocks ◽  
Nucleophilic Attack ◽  
Keto Esters ◽  
Key Enzyme

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze various transformations of amino acids at alpha, beta, and gamma positions. These versatile enzymes are prominently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products, and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2<i>S, </i>6<i>S</i>)-6-methyl pipecolate <b>1</b>, from the biosynthetic pathway of indole alkaloid citrinadin. The key enzyme CndF is PLP-dependent and catalyzes synthesis of (<i>S</i>)-2-amino-6-oxoheptanoate <b>3</b> that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives <b>1</b>. Biochemical characterization of CndF showed this enzyme performs gamma-elimination of <i>O</i>-acetyl L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new C<sub>gamma</sub>-C<sub>delta</sub> bond in <b>3 </b>and complete the gamma-substitution reaction. CndF displays substrate promiscuity towards different beta-keto carboxylate and esters. Using a recombinant <i>Aspergillus </i>strain expressing CndF and CndE, feeding various alkyl-beta-keto esters led to the biosynthesis of 6-substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.

scholarly journals A proposed methodology to standardize the determination of enzymic activities present in enzyme additives used in ruminant diets

2003 ◽  
Vol 83 (3) ◽  
pp. 559-568 ◽  
Author(s):  
D. Colombatto and K. A. Beauchemin
Keyword(s):  
Cell Walls ◽  
Production Efficiency ◽  
Biochemical Characterization ◽  
Nutrient Utilization ◽  
Plant Cell Walls ◽  
Poor Understanding ◽  
Ruminant Diets ◽  
Key Words Cellulase

There is increasing interest in using enzymes that degrade plant cell walls in ruminant diets to enhance production efficiency. Despite strong evidence from several studies suggesting a beneficial effect of enzyme supplementation on nutrient utilization and animal performance, overall the results have been somewhat inconsistent. One of the main problems faced by researchers is the lack of adequate biochemical characterization of the products used, which leads to a poor understanding of their mode of action. Of these biochemical characteristics, enzyme activities are the most important, but they are not always evaluated prior to use. Furthermore, as many arbitrary units of expression for these activities coexist, direct comparisons among studies are essentially impossible. In this paper, we propose a methodology that we feel accounts for the requirements of accuracy, simplicity and safety of use. In addition, a rationale for the standardization of the assays as a function of the conditions under which the enzymes are expected to act is presented. The standardization of these assays will benefit researchers, the feed industry, regulatory organizations, and ultimately the consumer, as it will result in the development of better, safer and more consistent enzyme additives for use in ruminant diets. Key words: Cellulase, enzyme additives, methodology, ruminants, xylanase

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