Deletion analysis of regions at the C-terminal part of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040

Structural Elucidation of the Cyclization Mechanism of α-1,6-Glucan by Bacillus circulans T-3040 Cycloisomaltooligosaccharide Glucanotransferase

“…Both constructs contained enzyme residues Ser-39 -Met-738 (molecular mass of 78 kDa), and the N-terminal signal peptide and the C-terminal variable region were omitted. Both protein forms were expressed in Escherichia coli BL21(DE3) cells, purified by His 6 tag nickel affinity chromatography and crystallized as reported previously (21,24). BcCITase-NH with a concentration of 7 mg/ml was crystallized by the sitting drop vapor diffusion method using a precipitant solution composed of 1.0 M sodium acetate trihydrate, 0.1 M HEPES, pH 7.5, 0.05 M cadmium sulfate hydrate, and 12%(v/v) glycerol, at 293 K. BcCITase-CH with a concentration of 20 mg/ml was crystallized using a precipitant solution composed of 1.6 M ammonium sulfate, 0.1 M MES, pH 6.5, and 10% (v/v) 1,4-dioxane.…”

“…Structural analysis using the ligandbound structures verified the catalytic nucleophile Asp-385 and also identified the catalytic acid/base Glu-453. Besides these GH66 core structural domains, BcCITase has two extra carbohydrate-binding module family 35 (CBM35) domains; one is inserted in the middle part of the conserved region, and this domain is common in CITases (BcCBM35-1); another is in the C-terminal region (BcCBM35-2) (20,21). The detailed function of these domains remains to be elucidated, although the presence of the first domain has been shown to stabilize the protein and to enhance the production of CI-8 (21).…”

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

“…Obviously, both PsDex and PsDex-CT formed CI-7 to CI-14 from dextran (Fig. 2C), whereas SmDex did not display the production of any cyclic sugars, implying that PsDex-carrying CIT-SR might contribute to the production of CIs, such as CITase (7). Because PsDR2-defective PsDex-CT produced CIs, the PsDR2 that was similar to CBM-6 did not contribute to cyclic sugar formation.…”

“…All dextranases reported so far are devoid of CIT-SR. CIT-SR, which forms carbohydrate-binding module , contributed to the CI formation of CITase (7). More recently, we resolved the three-dimensional structure of Dex from Streptococcus mutans (SmDex), which is truncated at the N-terminal variable region and C-VR (9,10).…”

“…4C, having about 20% homology at ␤-agarase from Saccharophagus degradans strain 2-40 (EMBL ID CP000282) (37) and ␣-agarase from Alteromonas agarilytica (GenBank number AAF26838.1) (38)), indicating that PsDex selected CBM-6 during protein evolution. Interestingly, Funane et al (7) predicted that CBM-35 at CIT-SR (BcCR1) catalytically contributes to CI production of CITase. This prediction prompted us to study CI production using PsDex and SmDex with and without CIT-SR, respectively.…”