title: The role of histidine residues in the catalytic act of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.
abstract: Our previous study on cyclomaltodextrin glucanotransferase (CGTase) by chemical modification implied the importance of one or two histidine residues in the cyclization reaction of the enzyme. Based on a computer modelled three-dimensional structure of the CGTase, five histidine residues were chosen as targets for the site-directed mutagenesis. The histidine residues 98, 140, 233 and 327 were replaced by aspartate and His-177 by proline using polymerase chain reaction-mediated techniques. The CGTase variants H98D, H140D, H233D and H327D resulted in a profound decrease in the cyclizing and amylolytic activities, while mutation H177P had little influence on the activities but affected the thermal stability and the width of the pH optimum. It is suggested that His-98 functions as (or as a significant part of) the subsite 2 for the binding of the substrate in CGTase and therefore H98D destabilizes the intermediate for cyclization, but does not markedly affect the hydrolytic reactions. Mutants H140D and H233D produced only minor amounts of alpha-cyclodextrin, did not exhibit substrate inhibition with maltotriose and showed non-Michaelis-Menten kinetics. It is proposed that the variants H140D, H233D and H327D cause steric hindrances near the active center, while mutation H177D has similar consequences on the same site spatially.
authors.affiliation: Department of Biochemistry, University of Turku, Finland.
authors.forename: P
authors.initials: P
authors.lastname: Mattsson
authors.forename: N
authors.initials: N
authors.lastname: Battchikova
authors.forename: K
authors.initials: K
authors.lastname: Sippola
authors.forename: T
authors.initials: T
authors.lastname: Korpela
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D005964:Glucosyltransferases / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction
chemical_list: D006639:Histidine; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase