TY - JOUR
T1 - Molecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase
AU - Albenne, Cécile
AU - Skov, Lars
AU - Mirza, Osman Asghar
AU - Gajhede, Michael
AU - Feller, Georges
AU - D'Amico, Salvino
AU - André, Gwénaëlle
AU - Potocki-Véronèse, Gabrielle
AU - van der Veen, Bart A
AU - Monsan, Pierre
AU - Remaud-Simeon, Magali
PY - 2004/1/2
Y1 - 2004/1/2
N2 - Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases that synthesizes an insoluble amylose-like polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with alpha-amylases. Exactly how this enzyme catalyzes the formation of alpha-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance (Arg415, subsite +4) and the correct positioning (Asp394 and Arg446, subsite +1) of acceptor molecules. On the other hand, Arg226 (subsites +2/+3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products.
AB - Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases that synthesizes an insoluble amylose-like polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with alpha-amylases. Exactly how this enzyme catalyzes the formation of alpha-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance (Arg415, subsite +4) and the correct positioning (Asp394 and Arg446, subsite +1) of acceptor molecules. On the other hand, Arg226 (subsites +2/+3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products.
KW - Amino Acid Sequence
KW - Amino Acid Substitution
KW - Binding Sites
KW - Conserved Sequence
KW - Glucosyltransferases
KW - Models, Molecular
KW - Mutagenesis, Site-Directed
KW - Neisseria
KW - Oligosaccharides
KW - Protein Conformation
KW - Recombinant Proteins
KW - Sequence Alignment
KW - Sequence Homology, Amino Acid
KW - Sucrose
U2 - 10.1074/jbc.M309891200
DO - 10.1074/jbc.M309891200
M3 - Journal article
C2 - 14570882
SN - 0021-9258
VL - 279
SP - 726
EP - 734
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
ER -