Yoshiaki Kitamura

Metabolism of Levoglucosan (1,6-Anhydro-α-d-glucopyranose) in Microorganisms

The metabolism of levoglucosan (1,6-anhydro-β-d-glucopyranose) in several microorganisms was studied. Although levoglucosan could be easily hydrolyzed to glucose with acid, all of the tested yeasts and fungi strains had no activity hydrolyzing it. On the other hand, the formation of glucose 6-phosphate from levoglucosan in the presence of Mg-ATP2− was observed in the reaction with the cell extracts of all levoglucosan-assimilating yeasts and fungi. The enzyme catalyzing this formation of glucose 6-phosphate was shown to be independent of the general hexokinases by an ion-exchange, chromatography or polyacrylamide gel electrophoresis followed by staining of the activities. These data showed that levoglucosan would be directly phosphorylated to glucose 6-phosphate with a specific enzyme, levoglucosan kinase, and then metabolized through the general glycolytic pathway.

Identification of Catalytic Amino Acids of Cyclodextran Glucanotransferase from Bacillus circulans T-3040

In glycoside hydrolase family 66 (see http://afmb.cnrs-mrs.fr/CAZY/), cyclodextran glucanotransferase (CITase) is the only transglycosylation enzyme, all the other family 66 enzymes being dextranases. To analyze the catalytic amino acids of CITase, we modified CITase chemically from the T-3040 strain of Bacillus circulans with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). EDC inactivated the enzyme by following pseudo-first order kinetics. In addition, the substrates of an isomaltooligosaccharide and a cyclodextran inhibited EDC-induced enzyme inactivation, implicating the carboxyl groups of CITase as the catalytic amino acids of the enzyme. When two conserved aspartic acid residues, Asp145 and Asp270, were replaced with Asn in T-3040 mature CITase, CIT-D270N was completely inactive, and CIT-D145N had reduced activity. The V max of CIT-D145N was 1% of that of wild-type CITase, whereas the K m of CIT-D145N was about the same as that of the wild-type enzyme. These findings indicate that Asp145 and Asp270 play an important role in the enzymatic reaction of T-3040 CITase.