Crystal Structure of the Polyextremophilic α-Amylase AmyB from Halothermothrix orenii: Details of a Productive Enzyme-Substrate Complex and an N Domain with a Role in Binding Raw Starch
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The gene for a membrane-bound, halophilic, and thermostable a-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove-the N-C groove-some 30 Šaway from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 Šresolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with a-cyclodextrin and maltoheptaose at 2.2 Šresolution, a maltotetraose molecule is bound in subsites - 1 to + 3, spanning the cleavage point at - 1/+ 1, with the - 1 glucosyl residue present as a 2So skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across - 1/+ 1 and ring distortion. We observe three methionine side chains that serve as "binding platforms" for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch. 頲008 Elsevier Ltd. All rights reserved.
Journal of Molecular Biology
Biochemistry and Cell Biology not elsewhere classified