Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins.

Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development.

Electrically Switchable Nanolever Technology for the Screening of Metal-Chelating Peptides in Hydrolysates.

Metal-chelating peptides (MCP) are considered as indirect antioxidants due to their capacity to inhibit radical chain reaction and oxidation. Here, we propose a new proof of concept for the screening of MCPs present in protein hydrolysates for valorizing their antioxidant properties by using the emerging time-resolved molecular dynamics technology, switchSENSE. This method unveils possible interactions between MCPs and immobilized nickel ions using fluorescence and electro-switchable DNA chips.

Diversity of Omega Glutathione Transferases in mushroom-forming fungi revealed by phylogenetic, transcriptomic, biochemical and structural approaches.

The Omega class of glutathione transferases (GSTs) forms a distinct class within the cytosolic GST superfamily because most of them possess a catalytic cysteine residue. The human GST Omega 1 isoform was first characterized twenty years ago, but it took years of work to clarify the roles of the human isoforms. Concerning the kingdom of fungi, little is known about the cellular functions of Omega glutathione transferases (GSTOs), although they are widely represented in some of these organisms.

Oxidized glutathione promotes association between eukaryotic translation elongation factor 1Bγ and Ure2p glutathione transferase from Phanerochaete chrysosporium.

The eukaryotic translation elongation factor 1Bγ (eEF1Bγ) is an atypical member of the glutathione transferase (GST) superfamily. Contrary to more classical GSTs having a role in toxic compound detoxification, eEF1Bγ is suggested to act as a scaffold protein, anchoring the elongation factor complex EF1B to the endoplasmic reticulum. In this study, we show that eEF1Bγ from the basidiomycete Phanerochaete chrysosporium is fully active as a glutathione transferase in vitro and undergoes conformational changes upon binding of oxidized glutathione.

Sinorhizobium meliloti YrbA binds divalent metal cations using two conserved histidines.

Sinorhizobium meliloti is a nitrogen-fixing bacterium forming symbiotic nodules with the legume Medicago truncatula. S. meliloti possesses two BolA-like proteins (BolA and YrbA), the function of which is unknown.

Affinity of chlordecone and chlordecol for human serum lipoproteins.

Chlordecone (CLD) is a chlorinated persistent organic pollutant (POP) whose presence despite the 1993 ban in agriculture areas has caused numerous public health concerns. CLD accumulates in the liver, and the CLD metabolite, chlordecol (CLD-OH) is found in bile, an important site of excretion for cholesterol transported to the liver via lipoproteins. Here, we studied the real-time molecular interaction between CLD and CLD-OH with human serum lipoproteins, LDL and HDL.

Molecular Dynamics to Elucidate the DNA-Binding Activity of AlpZ, a Member of the Gamma-Butyrolactone Receptor Family in .

Signaling molecule receptors play a central role in quorum sensing and in the coordination onset of specialized metabolite biosynthesis in due to their dual function in signal detection and gene expression control through DNA-binding in the promoter region of their target genes. In the biosynthetic gene cluster includes the signaling molecule receptor AlpZ that negatively regulates through a complex regulatory cascade the expression of key genes involved in the kinamycin antibiotic production until its cognate ligand, a yet unidentified signaling molecule, prompts its release from target promoters. Here we use an original molecular dynamics method to evaluate the DNA-binding properties of AlpZ to its target DNA sequence and the impact the signaling molecule has on the interaction.

Is there a role for tau glutathione transferases in tetrapyrrole metabolism and retrograde signalling in plants?

In plants, tetrapyrrole biosynthesis occurs in chloroplasts, the reactions being catalysed by stromal and membrane-bound enzymes. The tetrapyrrole moiety is a backbone for chlorophylls and cofactors such as sirohaems, haems and phytochromobilins. Owing to this diversity, the potential cytotoxicity of some precursors and the associated synthesis costs, a tight control exists to adjust the demand and the fluxes for each molecule.

Effect of nonenzymatic deamidation on the structure stability of Camelus dromedarius α-lactalbumin.

Camelid α-lactalbumin is the only known protein that can undergo nonenzymatic deamidation on two Asn residues. This leads to the generation of a mixture of unusual isoAsp and d-Asp residues that may impact health. The effect of deamidation on camel α-lactalbumin instability was investigated.

Crystal Structure of Saccharomyces cerevisiae ECM4, a Xi-Class Glutathione Transferase that Reacts with Glutathionyl-(hydro)quinones.

Glutathionyl-hydroquinone reductases (GHRs) belong to the recently characterized Xi-class of glutathione transferases (GSTXs) according to unique structural properties and are present in all but animal kingdoms. The GHR ScECM4 from the yeast Saccharomyces cerevisiae has been studied since 1997 when it was found to be potentially involved in cell-wall biosynthesis. Up to now and in spite of biological studies made on this enzyme, its physiological role remains challenging.

Interaction between dietary bioactive peptides of short length and bile salts in submicellar or micellar state.

Bile salts act as steroidal detergents in the gut, and could also interact with peptides and improve their bioavailability, although the mechanism is unclear. The occurrence of direct interaction between milk bioactive peptides, Ile-Asn-Tyr-Trp, Leu-Asp-Gln-Trp, and Leu-Gln-Lys-Trp, and different bile salts in the submicellar or micellar state was investigated by intrinsic fluorescence measurement and dynamic light scattering, above the critical micellar concentration, the latter being determined by isothermal titration calorimetry. The peptides form aggregates, spontaneously.

Insights into ascorbate regeneration in plants: investigating the redox and structural properties of dehydroascorbate reductases from Populus trichocarpa.

Dehydroascorbate reductases (DHARs), enzymes belonging to the GST superfamily, catalyse the GSH-dependent reduction of dehydroascorbate into ascorbate in plants. By maintaining a reduced ascorbate pool, they notably participate to H2O2 detoxification catalysed by ascorbate peroxidases (APXs). Despite this central role, the catalytic mechanism used by DHARs is still not well understood and there is no supportive 3D structure.

New Insights into the Proteolytic System of Streptococcus thermophilus: Use of Isracidin To Characterize Cell-Associated Extracellular Peptidase Activities.

The influence on the hydrolysis of isracidin of cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities in addition to protease PrtS of Streptococcus thermophilus strains was investigated. S. thermophilus LMD-9 (PrtS(+) phenotype) efficiently hydrolyzed the isracidin mainly through the PrtS activity, whereas strain CNRZ1066 (PrtS(-) phenotype) and two mutant strains LMD-9-ΔprtS and LMD-9-ΔprtS-ΔhtrA also displayed substrate hydrolysis, but different from that of the wild type strain LMD-9.

Identification by FT-ICR-MS of Camelus dromedarius α-lactalbumin variants as the result of nonenzymatic deamidation of Asn-16 and Asn-45.

Nonenzymatic deamidation of asparaginyl residues can occur spontaneously under physiological conditions principally when a glycyl residue is at the carboxyl side of Asn and leads to formation of aspartyl and isoaspartyl residues. This modification can change the biological activity of proteins or peptides and trigger an auto-immune response. The α-lactalbumins of members of the Camelidae family are the only of described α-lactalbumins that carry two AsnGly sequences.

Determination of reducing power and metal chelating ability of antioxidant peptides: revisited methods.

The purpose of this study was to improve two common tests used for antioxidant capacity measurements, i.e. the reducing power and chelating ability measurements, for appropriate comparisons between the molecules tested and chosen references, as the usual methods are often performed in a qualitative way rather than a quantitative way.

A single Sfp-type phosphopantetheinyl transferase plays a major role in the biosynthesis of PKS and NRPS derived metabolites in Streptomyces ambofaciens ATCC23877.

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis.

Surface plasmon resonance analysis of the binding mechanism of pharmacological and peptidic inhibitors to human somatic angiotensin I-converting enzyme.

Somatic angiotensin I-converting enzyme (ACE) possesses two catalytic domains and plays a major role in the regulation of blood pressure, thus representing a therapeutic target for the treatment of hypertension. We present a comprehensive surface plasmon resonance (SPR) study of the interaction of human somatic ACE with the pharmacological inhibitors captopril and lisinopril, the bradykinin potentiating peptide BPP-11b, and the food peptidic inhibitors from bovine αs2-casein, F(174)ALPQYLK(181) and F(174)ALPQY(179). SPR binding curves recorded with the high potency inhibitors captopril, lisinopril, and BPP-11b were evaluated both by regression analysis and by kinetic distribution analysis.

Hydrolysis of milk-derived bioactive peptides by cell-associated extracellular peptidases of Streptococcus thermophilus.

The trend to confer new functional properties to fermented dairy products by supplementation with bioactive peptides is growing in order to encounter the challenge of health-promoting foods. But these functional ingredients have not to be hydrolysed by proteases of bacteria used in the manufacture of these products. One of the two yoghurt bacteria, Streptococcus thermophilus, has long been considered as weakly proteolytic since its only cell wall-associated subtilisin-like protease, called PrtS, is not always present.

Lactoferrin and its hydrolysate bind directly to the oleate-activated form of the lipolysis stimulated lipoprotein receptor.

The hepatic removal of triglyceride-rich chylomicrons during the postprandial phase represents an important step towards determining the bioavailability of dietary lipids amongst the peripheral tissues. Indeed, elevated postprandial lipemia is often associated with obesity and increased risk of coronary heart disease. The milk protein, lactoferrin, has been shown to inhibit hepatic chylomicron remnant removal by the liver, resulting in increased postprandial lipemia.

Binding of divalent metal ions to 1-25 β-caseinophosphopeptide: an isothermal titration calorimetry study.

To better understand the mechanism of metal ion transport through the gastrointestinal tract to their absorption sites, isothermal titration calorimetry (ITC) was used to investigate the binding of dicationic metals to β-CN(1-25)4P, a β-casein tetraphosphorylated peptide. ITC technology was found suitable for studying weak bonds between metal ions and phosphopeptides and provided a direct means of thermodynamic and stoichiometric characterisation of complex formation. Thus, one mole of β-CN(1-25)4P binds two moles of Ca(2+), Mg(2+) or Zn(2+) under experimental conditions close to those of the ileum (pH 8, 37°C), with rather low binding affinity constants (K=4900-11,200M(-1)).

Identification of phosphorylation sites of equine beta-casein isoforms.

Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P-7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion-exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro-column.

The primary structure of a low-Mr multiphosphorylated variant of beta-casein in equine milk.

Highly phosphorylated casein with a low molecular mass was isolated from Haflinger mare's milk by RP-HPLC. It accounts for 4.0% of the casein content.

Determination of the phosphorylation level and deamidation susceptibility of equine beta-casein.

beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.

Functional angucycline-like antibiotic gene cluster in the terminal inverted repeats of the Streptomyces ambofaciens linear chromosome.

Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the beta-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS.