Identification of key amino acid residues in Neisseria polysaccharea amylosucrase.
Amylosucrase from Neisseria polysaccharea catalyzes the synthesis of an amylose-like polymer from sucrose. Sequence alignment revealed that it belongs to the glycoside hydrolase family 13. Site-directed mutagenesis enabled the identification of functionally important amino acid residues located at the active center. Asp-294 is proposed to act as the catalytic nucleophile and Glu-336 as general acid base catalyst in amylosucrase. The conserved Asp-401, His-195 and His-400 residues are critical for the enzymatic activity. These results provide strong support for the predicted close structural and functional relationship between the sucrose-glucosyltransferases and enzymes of the alpha-amylase family.
Sarçabal
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P
Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, UR INRA 792, INSA, 135 Avenue de Rangueil, 31 077 Toulouse Cedex 4, France.
Remaud-Simeon
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M
Willemot
R
R
Potocki de Montalk
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G
Svensson
B
B
Monsan
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P
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid; D006639:Histidine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009343:Neisseria / Q000201:enzymology*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D000596:Amino Acids; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D005964:Glucosyltransferases; C019700:amylosucrase