Identification of catalytic amino acids of cyclodextran glucanotransferase from Bacillus circulans T-3040

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2006-08, Vol.70 (8), p.1947-1953
• Main Authors: Yamamoto, T.(National Food Research Inst, Tsukuba, Ibaraki (Japan)), Terasawa, K, Kim, Y.M, Kimura, A, Kitamura, Y, Kobayashi, M, Funane, K
• Format: Article
• Language: English
• Subjects: ACIDE AMINE; ACTIVIDAD CATALITICA; ACTIVITE CATALYTIQUE; Amino Acid Sequence; AMINO ACIDS; Amino Acids - chemistry; AMINOACIDOS; aspartate residue; Bacillus - enzymology; BACILLUS CIRCULANS; Biological and medical sciences; CARIE DENTAIRE; CARIES DENTAL; Catalysis; CATALYTIC ACTIVITY; catalytic amino acid; Catalytic Domain - genetics; cyclodextran; cyclodextran glucanotransferase; DENTAL CARIES; DEXTRANAS; DEXTRANE; DEXTRANS; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic; GLICOSILTRANSFERASAS; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - isolation & purification; GLYCOSYLTRANSFERASE; GLYCOSYLTRANSFERASES; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.60105

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 Vsub(max) of CIT-D145N was 1% of that of wild-type CITase, whereas the Ksub(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.