chitotetrose and chitotriose

Cellulose is a linear homopolymer of beta 1-4 linked glucose residues. Chitin is similar to cellulose in structure, and can be described as cellulose with the hydroxyl group on the C2 carbon replaced by an acetylamine group. Both cellulose and chitin form tightly packed, extensively hydrogen-bonded micro-fibrils. Up to now, binding of cellulase catalytic domains (CDs) to chitin has not been reported. In this article, binding of the CDs of Thermobifida fusca Cel6A, Cel6B, Cel48A, Cel5A, and Cel9A to alpha-chitin was investigated. The CDs of endocellulases, Cel6A and Cel5A did not bind to alpha-chitin; one exocellulase, Cel48A CD bound alpha-chitin moderately well; and the exocellulase Cel6B CD and the processive endocellulase Cel9A CD bound extremely tightly to alpha-chitin. Only mutations of Cel6B W329C, W332A and G234S and Cel9A Y206F, Y206S and D261A/R378K caused weaker binding to alpha-chitin than wild-type, and all these mutations were of residues near the catalytic center. One mutant enzyme, Cel9A D261A/R378K had weak chitinase activity, but no soluble products were detected. Chitotriose and chitotetraose were docked successfully to the catalytic cleft of Cel9A. In general, the positioning of the sugar residues in the model structures matched the cellooligosaccharides in the X-ray structure. Our results show that the binding of chitin by a cellulase can provide additional information about its binding to cellulose.

Topics: Actinomycetales; Amino Acid Substitution; Binding Sites; Catalytic Domain; Cellulase; Cellulose; Chitin; Models, Chemical; Mutagenesis, Site-Directed; Oligosaccharides; Protein Binding; Substrate Specificity; Trisaccharides

A specific substrate binding protein is located within the membrane of Streptomyces olivaceoviridis mycelia. After Triton extraction of the membrane, two forms of the protein (46.0 kD and 47.5 kD) were purified to apparent homogeneity by consecutive anionic exchange chromatographies. The results of competition interacted with N-acetylglucosamine and chitin oligomers (C2 to C6), but not with cellobiose nor glucose. Using surface plasmon resonance, the kinetic parameters of the 46 kD form of the binding protein were determined. This protein showed a very high affinity for N-acetylglucosamine (K(d) = 8.29 x 10(-9) mol/L) and for chitobiose (K(d) = 3.81 X 10(-6) mol/L), and the lowest one was for chitotriose (K(d) = 1.95 X 10(-5) mol/L). Comparisons of the dissociation and association rate constants indicated that the interaction of this protein with each ligand was controlled by the association rate. N terminal sequence indicated that this protein might belong to an ABC transporter system.

Topics: Acetylglucosamine; Amino Acid Sequence; Bacterial Proteins; Binding, Competitive; Chitin; Dimerization; Disaccharides; Kinetics; Molecular Sequence Data; Molecular Weight; Oligosaccharides; Protein Binding; Sequence Analysis, Protein; Streptomyces; Trisaccharides

Rhizobium bacteria synthesize signal molecules called Nod factors that elicit responses in the legume root during nodulation. Nod factors, modified N-acylated beta-(1,4)-N-acetylglucosamine, are synthesized by the nodulation (nod) gene products. We tested the ability of three Sinorhizobium meliloti nod gene products to modify Nod factor analogs with thio linkages instead of O-glycosidic bonds in the oligosaccharide backbone.

Topics: Acyltransferases; Amidohydrolases; Bacterial Proteins; Oligosaccharides; Sinorhizobium meliloti; Sulfotransferases; Trisaccharides

Induction and repression of a gene for chitinase (chiA) in Streptomyces lividans was investigated using a catechol 2,3-dioxygenase gene (xylE) as the reporter gene. Of various substrates examined, expression of the promoter (PchiA) was observed after a delay when colloidal chitin or small chitin-oligosaccharides were added to the medium. N-acetylglucosamine completely repressed the chiA promoter. The duration of the delay in expression of PchiA differed with the inducer used, with chitobiose inducing the activity most rapidly. The minimum concentration of chitobiose needed for induction was 1 microM. It appears, therefore, that an efficient inducer of the gene for chitinase in S. lividans is chitobiose.

Topics: Catechol 2,3-Dioxygenase; Chitinases; Dioxygenases; Disaccharides; Enzyme Induction; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Oligosaccharides; Oxygenases; Promoter Regions, Genetic; Streptomyces; Trisaccharides

The mechanism underlying molecular recognition of lectins was elucidated by a novel solid phase binding assay system based on surface plasmon resonance. When the apparent affinities of interactions between chitooligosaccharides and wheat germ agglutinin were compared between lectin-immobilized and oligosaccharide-immobilized assay systems, the affinity constants (Ka) calculated for the former system were in good agreement with the previously reported values measured in solution. On the other hand, in the latter system, the calculated Ka could be more than 10,000 times higher than the values in solution at lower lectin concentrations. To elucidate the reason for this, we systematically investigated the effects of the oligosaccharide immobilized density and the lectin valence on the apparent affinity in the oligosaccharide-immobilized assay system. Both the apparent association (k[ass]) and dissociation rate constants (k[diss]) showed a tendency to decrease as the oligosaccharide density increased. This effect was most remarkable for the interaction possessing an extremely fast intrinsic k(ass). Oligomerization of lectin enhanced the avidity due to a significant reduction in k(diss). These phenomena could be explained by considering the nonhomogeneous conditions under which binding occurred. The reaction in a nonhomogeneous state is limited by the mass transport effect, and the effect of rebinding becomes so large that it cannot be disregarded. These findings are the first to demonstrate the importance of the mass transport effect in modulating the affinity of lectin for oligosaccharides on a solid phase surface.

Topics: Amino Sugars; Biosensing Techniques; Biotinylation; Chemical Phenomena; Chemistry, Physical; Chitin; Disaccharides; Kinetics; Lectins; Molecular Weight; Oligosaccharides; Phytohemagglutinins; Plant Lectins; Ribosome Inactivating Proteins; Trisaccharides; Wheat Germ Agglutinins