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Crystal structure of the polyextremophilic alpha-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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2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 378, no 4, 852-870 p.Article in journal (Refereed) Published
Abstract [en]

The gene for a membrane-bound, halophilic, and thermostable alpha-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 angstrom away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 angstrom resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with alpha-cyclodextrin and maltoheptaose at 2.2 angstrom resolution, 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 S-2(o) 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.

Place, publisher, year, edition, pages
2008. Vol. 378, no 4, 852-870 p.
Keyword [en]
alpha-amylase, N domain, enzyme-substrate complex, polyextremophilic, methionine interaction, complete nucleotide-sequence, bacillus-licheniformis, angstrom, resolution, 3-dimensional structure, xylose isomerase, active-sites, protein, stability, family, mechanism
Identifiers
URN: urn:nbn:se:kth:diva-17563DOI: 10.1016/j.jmb.2008.02.041ISI: 000256180100008Scopus ID: 2-s2.0-41949118483OAI: oai:DiVA.org:kth-17563DiVA: diva2:335607
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved

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Divne, Christina

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