tailieunhanh - Báo cáo khoa học: nsights into the reaction mechanism of glycosyl hydrolase family 49 Site-directed mutagenesis and substrate preference of isopullulanase

Aspergillus nigerisopullulanase (IPU) is the only pullulan-hydrolase inglycosyl hydrolase (GH) family 49 anddoes not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes. To investigate the common catalyticmechanismofGHfamily 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three Asp and two Glu). | Eur. J. Biochem. 271 4420-4427 2004 FEBS 2004 doi Insights into the reaction mechanism of glycosyl hydrolase family 49 Site-directed mutagenesis and substrate preference of isopullulanase Hiromi Akeboshi1 Takashi Tonozuka1 Takaaki Furukawa1 Kazuhiro Ichikawa1 Hiroyoshi Aoki1 2 Akiko Shimonishi1 Atsushi Nishikawa1 and Yoshiyuki Sakano1 1 Department of Applied Biological Science Tokyo University of Agriculture and Technology Fuchu Tokyo Japan 2Fuence Co. Shibuya Tokyo Japan 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 wildtype 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 b-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 .

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