Crystal structure of AmyB, an alpha-amylase from Halothermothrix orenii, and comparison with its homologs

Abstract

We have determined, by means of molecular replacement, the crystal structure of the halotolerant and thermostable alpha-amylase AmyB from Halothermothrix orenii at 2.3 Angstroms resolution. In addition, the structures of AmyB in complex with a nonasaccharide resulting from transglycosylation of the inhibitor acarbose at 1.35 Angstroms resolution, and the 2.2 Angstroms structure of the enzyme in complex with hydrolysis products of maltoheptaose have been determined. The crystal structures of AmyB complexes have also made it possible to identify a novel binding site for raw starch formed by the N-terminal domain and the rest of the molecule, the N-C groove. Results from starch-binding studies using full-length AmyB and a truncated mutant lacking the N domain show that the presence of the N domain enhances binding to the insoluble substrate. Results presented in this thesis show that AmyB is indeed unique compared with other N1-amylases in that it is membrane bound, monomeric, and carries an N-terminal domain between the membrane linker and domain A that forms a large groove for binding of raw starch.