Crystal Structures of Amylosucrase from Neisseria polysaccharea in Complex with d-Glucose and the Active Site Mutant Glu328Gln in Complex with the Natural Substrate Sucrose

• Published in: Biochemistry (Easton) 2001-07, Vol.40 (30), p.9032-9039
• Main Authors: Mirza, Osman, Skov, Lars K, Remaud-Simeon, Magali, Potocki de Montalk, Gabrielle, Albenne, Cecile, Monsan, Pierre, Gajhede, Michael
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Binding Sites - genetics; Carbohydrate Conformation; Crystallization; Crystallography, X-Ray; glucose; Glucose - chemistry; Glucose - metabolism; Glucosyltransferases - chemistry; Glucosyltransferases - metabolism; Glutamic Acid - genetics; Glutamine - genetics; Hydrogen Bonding; Ligands; Macromolecular Substances; Models, Molecular; Neisseria - enzymology; Neisseria - genetics; Neisseria polysaccharea; Point Mutation; Substrate Specificity - genetics; sucrose; Sucrose - chemistry; Sucrose - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010706l

The structure of amylosucrase from Neisseria polysaccharea in complex with β-d-glucose has been determined by X-ray crystallography at a resolution of 1.66 Å. Additionally, the structure of the inactive active site mutant Glu328Gln in complex with sucrose has been determined to a resolution of 2.0 Å. The d-glucose complex shows two well-defined d-glucose molecules, one that binds very strongly in the bottom of a pocket that contains the proposed catalytic residues (at the subsite −1), in a nonstrained 4C1 conformation, and one that binds in the packing interface to a symmetry-related molecule. A third weaker d-glucose-binding site is located at the surface near the active site pocket entrance. The orientation of the d-glucose in the active site emphasizes the Glu328 role as the general acid/base. The binary sucrose complex shows one molecule bound in the active site, where the glucosyl moiety is located at the α-amylase −1 position and the fructosyl ring occupies subsite +1. Sucrose effectively blocks the only visible access channel to the active site. From analysis of the complex it appears that sucrose binding is primarily obtained through enzyme interactions with the glucosyl ring and that an important part of the enzyme function is a precise alignment of a lone pair of the linking O1 oxygen for hydrogen bond interaction with Glu328. The sucrose specificity appears to be determined primarily by residues Asp144, Asp394, Arg446, and Arg509. Both Asp394 and Arg446 are located in an insert connecting β-strand 7 and α-helix 7 that is much longer in amylosucrase compared to other enzymes from the α-amylase family (family 13 of the glycoside hydrolases).