title: Insights into the reaction mechanism of glycosyl hydrolase family 49. Site-directed mutagenesis and substrate preference of isopullulanase.
abstract: Aspergillus niger isopullulanase (IPU) is the only pullulan-hydrolase in glycosyl hydrolase (GH) family 49 and does not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes. To investigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three Asp and two Glu). Homology modelling of IPU was also carried out based on the structure of Penicillium minioluteum dextranase, and the result showed that Asp353, Glu356, Asp372, Asp373 and Trp402, whose substitutions resulted in the reduction of activity for both pullulan and panose, were predicted to be located in the negatively numbered subsites. Three Asp-mutated enzymes, D353N, D372N and D373N, lost their activities, indicating that these residues are candidates for the catalytic residues of IPU. The W402F enzyme significantly reduced IPU activity, and the Km value was sixfold higher and the k0 value was 500-fold lower than those for the wild-type enzyme, suggesting that Trp402 is a residue participating in subsite -1. Trp31 and Glu273, whose substitutions caused a decrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal and beta-helical domains. The substrate preference of the negatively numbered subsites of IPU resembles that of GH family 49 dextranases. These findings suggest that IPU and the GH family 49 dextranases have a similar catalytic mechanism in their negatively numbered subsites in spite of the difference of their substrate specificities.
authors.affiliation: Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Akeboshi
authors.forename: Takashi
authors.initials: T
authors.lastname: Tonozuka
authors.forename: Takaaki
authors.initials: T
authors.lastname: Furukawa
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Ichikawa
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Aoki
authors.forename: Akiko
authors.initials: A
authors.lastname: Shimonishi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishikawa
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Sakano
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes; D008320:Maltose / Q000031:analogs & derivatives / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005936:Glucans; D007527:Isoenzymes; D011994:Recombinant Proteins; C008763:panose; D008320:Maltose; C009109:pullulan; D006026:Glycoside Hydrolases; C047371:isopullulanase; D005947:Glucose