title: Identification of catalytic amino acids of cyclodextran glucanotransferase from Bacillus circulans T-3040.
abstract: 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.
authors.affiliation: National Food Research Institute, Tsukuba, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Kazue
authors.initials: K
authors.lastname: Terasawa
authors.forename: Young-Min
authors.initials: YM
authors.lastname: Kim
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kitamura
authors.forename: Mikihiko
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Kazumi
authors.initials: K
authors.lastname: Funane
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C087517:cycloisomaltooligosaccharide glucanotransferase