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

VL - 279

SP - 726

EP - 734

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 1

ER -