In order to reveal the origin of carbohydrate recognition specificity of human lysozyme by clarifying the difference in the binding mode of ligands in the active site, the inactivation of human lysozyme by 2',3'-epoxypropyl beta-glycoside derivatives of the disaccharides, N,N'-diacetylchitobiose [GlcNAc-beta-(1-->4)-GlcNAc] and N-acetyllactosamine [Gal-beta-(1-->4)-GlcNAc], was investigated and the three-dimensional structures of the affinity-labeled enzymes were determined by X-ray crystallography at 1.7 A resolution. Under the conditions comprising 2.0 x 10(-3) M labeling reagent and 1.0 x 10(-5) M human lysozyme at pH 5.4, 37 degrees C, the reaction time required to reduce the lytic activity against Micrococcus luteus cells to 50% of its initial activity was lengthened by 3.7 times through the substitution of the nonreducing end sugar residue, GlcNAc to Gal. The refined structure of human lysozyme labeled by 2',3'-epoxypropyl beta-glycoside derivatives of N,N'-diacetylchitobiose (HL/NAG-NAG-EPO complex) indicated that the interaction mode of the N,N'-diacetylchitobiose moiety in substites B and C in this study was essentially the same as in the case of the complex of human lysozyme with the free ligand. On the other hand, the hydrogen-bonding pattern and the stacking interaction at subsite B were remarkably different between the HL/NAG-NAG-EPO complex and human lysozyme labeled by the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine (HL/GAL-NAG-EPO complex). The reduced number of possible hydrogen bonds as well as the less favorable stacking between the side chain of Tyr63 in human lysozyme and the galactose residue in the HL/GAL-NAG-EPO complex reasonably explained the less efficient ability of the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine as compared to that of N,N'-diacetylchitobiose as an affinity labeling reagent toward human lysozyme.
A stereoselective and efficient total synthesis of optically active tetrodotoxin (TTX) is described. A polyfunctionalized key cyclitol compound containing branched-chains for the synthesis of TTX was prepared from D-glucose employing the Henry reaction (Nitro aldol reaction) as the key transformation. Stereoselective construction of the alpha-azido-aldehyde branched-chain was achieved via the key spiro alpha-chloroepoxide intermediate.
The synergism between apolar and polar interactions in the carbohydrate recognition by human lysozyme (HL) was probed by site-directed mutagenesis and affinity labeling. The three-dimensional structures of the Tyr63-->Leu mutant HL labeled with 2',3'-epoxypropyl beta-glycoside of N,N'-diacetylchitobiose (L63-HL/NAG-NAG-EPO complex) and the Asp102-->Glu mutant HL labeled with the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine were revealed by X-ray diffraction at 2.23 and 1.96 A resolution, respectively. Compared to the wild-type HL labeled with the 2', 3'-epoxypropyl beta-glycoside of N,N'-diacetylchitobiose, the N-acetylglucosamine residue at subsite B of the L63-HL/NAG-NAG-EPO complex markedly moved away from the 63rd residue, with substantial loss of hydrogen-bonding interactions. Evidently, the stacking interaction with the aromatic side chain of Tyr63 is essential in positioning the N-acetylglucosamine residue in the productive binding mode. On the other hand, the position of the galactose residue in subsite B of HL is almost unchanged by the mutation of Asp102 to Glu. Most hydrogen bonds, including the one between the carboxylate group of Glu102 and the axial 4-OH group of the galactose residue, were maintained by local movement of the backbone from residues 102-104. In both structures, the conformation of the disaccharide was conserved, reflecting an intrinsic conformational rigidity of the disaccharides. The structural analysis suggested that CH-pi interactions played an important role in the recognition of the carbohydrate residue at subsite B of HL.
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