Molecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase.
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.
Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, INSA, 31077 Toulouse Cedex 4, France.
Cécile
C
Albenne
Lars K
LK
Skov
Osman
O
Mirza
Michael
M
Gajhede
Georges
G
Feller
Salvino
S
D'Amico
Gwénaëlle
G
André
Gabrielle
G
Potocki-Véronèse
Bart A
BA
van der Veen
Pierre
P
Monsan
Magali
M
Remaud-Simeon
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D017124:Conserved Sequence; D005964:Glucosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009343:Neisseria / Q000201:enzymology*; D009844:Oligosaccharides / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000378:metabolism
D009844:Oligosaccharides; D011994:Recombinant Proteins; D013395:Sucrose; D005964:Glucosyltransferases; C019700:amylosucrase