No abstract

Levoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-d-glucose by LgdB2; next, LgdC reduces 3-keto-d-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

We designed and synthesized three tetravalent sialo-glycoclusters that had different separations between the terminal sialic acids and the linking carboxy groups of the ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetate scaffold to serve as ligands for the sialic acid-binding lectin Sambucus sieboldiana agglutinin (SSA). The interaction between each glycocluster and SSA was characterized by hemagglutination inhibition, quantitative precipitation, and double-diffusion assays. For the precipitation assays, the precipitin curves indicated that the ligands and SSA bound in either a 1:1 or a 1:2 ratio, i.e., stoichiometrically. The strong interactions of these sialo-glycoclusters with SSA could be ascribed to a combination of multivalency and spacer effects. We also assessed the nature of the ligand-SSA complexes by isothermal titration calorimetry and dynamic light scattering. The results of those experiments indicated that formation of intermolecular complexes occurred at less than stoichiometric ratios of ligand to SSA concentrations and that, as the concentrations of the ligands increased, larger cross-linked aggregates formed. Large aggregates that were present concurrently with visible precipitation and with a particle size centered at ~600 to 800 nm were identified by dynamic light scattering.