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.

The structure of amylosucrase from Neisseria polysaccharea in complex with beta-D-glucose has been determined by X-ray crystallography at a resolution of 1.66 A. Additionally, the structure of the inactive active site mutant Glu328Gln in complex with sucrose has been determined to a resolution of 2.0 A. 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 (4)C(1) 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 alpha-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 beta-strand 7 and alpha-helix 7 that is much longer in amylosucrase compared to other enzymes from the alpha-amylase family (family 13 of the glycoside hydrolases).

Identification of key amino acid residues in Neisseria polysaccharea amylosucrase.

Amylosucrase from Neisseria polysaccharea catalyzes the synthesis of an amylose-like polymer from sucrose. Sequence alignment revealed that it belongs to the glycoside hydrolase family 13. Site-directed mutagenesis enabled the identification of functionally important amino acid residues located at the active center. Asp-294 is proposed to act as the catalytic nucleophile and Glu-336 as general acid base catalyst in amylosucrase. The conserved Asp-401, His-195 and His-400 residues are critical for the enzymatic activity. These results provide strong support for the predicted close structural and functional relationship between the sucrose-glucosyltransferases and enzymes of the alpha-amylase family.

Amylosucrase from Neisseria polysaccharea: novel catalytic properties.

Amylosucrase is a glucosyltransferase that synthesises an insoluble alpha-glucan from sucrose. The catalytic properties of the highly purified amylosucrase from Neisseria polysaccharea were characterised. Contrary to previously published results, it was demonstrated that in the presence of sucrose alone, several reactions are catalysed, in addition to polymer synthesis: sucrose hydrolysis, maltose and maltotriose synthesis by successive transfers of the glucosyl moiety of sucrose onto the released glucose, and finally turanose and trehalulose synthesis - these two sucrose isomers being obtained by glucosyl transfer onto fructose. The effect of initial sucrose concentration on initial activity demonstrated a non-Michaelian profile never previously described.

Characterisation of the activator effect of glycogen on amylosucrase from Neisseria polysaccharea.

Amylosucrase produces an insoluble alpha-1,4-linked glucan from sucrose, releasing fructose. In addition to polymerisation, in the presence of sucrose as sole substrate, amylosucrase catalyses sucrose hydrolysis and oligosaccharide synthesis in significant proportions. The effects of both glycogen acceptor and sucrose concentrations on the reactions catalysed by the highly purified amylosucrase from Neisseria polysaccharea were investigated. Sucrose hydrolysis decreased strongly with the increase of the concentration of glycogen, as did oligosaccharide synthesis, by glucose transfer onto glucose and fructose. The glucosyl units consumed were then preferentially used for elongation of glycogen chains. The study of the kinetic behaviour of amylosucrase revealed a strong, sucrose concentration dependent activator effect of glycogen. This activation was decreased at high sucrose concentration. The optimal sucrose concentrations increased with glycogen concentration, suggesting competition between sucrose and glycogen, and the presence of a second non-catalytic acceptor binding site which could bind various acceptors (glucose, maltose, glycogen) and also sucrose.

Crystallization and preliminary X-ray studies of recombinant amylosucrase from Neisseria polysaccharea.

Recombinant amylosucrase from Neisseria polysaccharea was crystallized by the vapour-diffusion procedure in the presence of polyethylene glycol 6000. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 95.7, b = 117.2, c = 62.1 A, and diffract to 1.6 A resolution. A p-chloromercuribenzene sulfonate (pcmbs) derivative has been identified and a selenomethionine-substituted protein has been produced and crystallized.