Biomedical subjects

S Drouillard

Publications and source records attributed to S Drouillard.

4 recordsLinked to original sources

PubMed1999

Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures.

Rhizobial lipo-chitooligosaccharides (LCOs) are signaling molecules involved in host-range recognition for the establishment of the symbiosis with leguminous plants. The major LCO of Rhizobium meliloti, the symbiont of Medicago plants contains four or five N-acetylglucosamines, O-acetylated and N-acylated with a C16:2 fatty acid on the terminal nonreducing sugar and O-sulfated on the reducing sugar. In this paper, the ligand specificity of a high-affinity binding site (Nod factor binding site 2 or NFBS2), enriched in a plasma membrane-enriched fraction of Medicago cell suspension cultures, is reported. By using chemically synthesized LCOs, the role of structural elements, important for symbiotic activities, as recognition motifs for NFBS2 was determined. The results show that the substitutions on the nonreducing sugar of the LCOs (the O-acetate group, the fatty acid, and the hydroxyl group on the C4 of the sugar) are determinants for high-affinity binding to NFBS2. In contrast, the sulfate group, which is necessary for all biological activities on Medicago, is not discriminated by NFBS2. However, the reducing sugar of the LCO seems to interact with NFBS2, because ligand binding is affected by the reduction of the free anomeric carbon and depends on the number of N-acetyl glucosamine residues. These results suggest that the recognition of the LCOs by NFBS2 is mediated by structural elements in both the lipid and oligosaccharidic moities, but not by the sulfate group.

Journal Article↗

PubMed1997

Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation.

The stereochemistry of the reaction catalysed by Serratia marcescens chitobiase was determined by HPLC separation of the anomers of N-acetylglucosamine produced during the hydrolysis of p-nitrophenyl N-acetyl-beta-d-glucosaminide (PNP-GlcNAc). In the early stages of the reaction, the beta-anomer was found to prevail, whereas the alpha-anomer dominated at mutarotation equilibrium. This established that chitobiase hydrolyses glycosidic bonds with overall retention of the anomeric configuration. Chitobiase-catalysed hydrolysis of PNP-GlcNAc was competitively inhibited by a series of chito-oligosaccharides (degree of polymerization 2-5) that were selectively de-N-acetylated at their non-reducing end. The results are in accord with the participation of the acetamido group at C-2 of the substrate in the catalytic mechanism of chitobiase and related enzymes.

Acetylation↗

PubMed1997

Gram-scale synthesis of recombinant chitooligosaccharides in Escherichia coli.

Cultivation of Escherichia coli harbouring heterologous genes of oligosaccharide synthesis is presented as a new method for preparing large quantities of high-value oligosaccharides. To test the feasibility of this method, we successfully produced in high yield (up to 2.5 g/L) penta-N-acetyl-chitopentaose (1) and its deacetylated derivative tetra-N-acetyl-chitopentaose (2) by cultivating at high density cells of E. coli expressing nodC or nodBC genes (nodC and nodB encode for chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively). These two products were easily purified by charcoal adsorption and ion-exchange chromatography. One important application of compound 2 could be its utilisation as a precursor for the preparation of synthetic nodulation factors by chemical acylation.

Acetylation↗

PubMed1997

A bifunctionalized fluorogenic tetrasaccharide as a substrate to study cellulases.

Cellulases are usually classified as endoglucanases and cellobiohydrolases, but the heterogeneity of cellulose, in terms of particle size and crystallinity, has always represented a problem for the biochemical characterization of the enzymes. The synthesis of a bifunctionalized tetrasaccharide substrate suitable for measuring cellulase activity by resonance energy transfer is described. The substrate, which carries a 5-(2-aminoethylamino)-1-naphthalenesulfonate group on the non-reducing end and an indolethyl group on the reducing end, was prepared from beta-lactosyl fluoride and indolethyl beta-cellobioside by a chemoenzymatic approach using the transglycosylating activity of endoglucanase I of Humicola insolens as the key step. The bifunctionalized substrate has been used for the determination of the catalytic constants of H. insolens endoglucanase I and cellobiohydrolases I and II; this substrate could be of general use to measure the kinetic constants of cellulases able to act on oligomers of degree of polymerization <5. The data also provide evidence that cellobiohydrolases I and II are able to degrade an oligosaccharide substrate carrying non-carbohydrate substituents at both ends.

Carbohydrate Conformation↗