Crystal structures of amylosucrase from Neisseria polysaccharea in complexwith D-glucose and the active site mutant Glu328Gln in complex with the natural substrate sucrose

The structure of amylosucrase from Neisseria polysaccharea in complex with beta -D-glucose has been determined by X-ray crystallography at a resolutio n of 1.66 Angstrom. Additionally, the structure of the inactive active site mutant Glu328Gln in complex with sucrose has been determined to a resoluti on of 2.0 A. The D-glucose complex shows two well-defined D-glucose molecul es, one that binds very strongly in the bottom of a pocket that contains th e proposed catalytic residues (at the subsite -1), in a nonstrained C-4(1) conformation, and one that binds in the packing interface to a symmetry-rel ated molecule. A third weaker D-glucose-binding site is located at the surf ace 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 alpha -amylase -1 position and the fr uctosyl ring occupies subsite +1. Sucrose effectively blocks the only visib le access channel to the active site. From analysis of the complex it appea rs that sucrose binding is primarily obtained through enzyme interactions w ith 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 bo nd interaction with Glu328. The sucrose specificity appears to be determine d primarily by residues Asp144, Asp394, Arg446, and Arg509. Both Asp394 and Arg446 are located in an insert connecting beta -strand 7 and alpha -helix 7 that is much longer in amylosucrase compared to other enzymes from the a lpha -amylase family (family 13 of the glycoside hydrolases).