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Crystal structure o...
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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Tan, Tien-Chye (författare)
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Mijts, Benjamin N. (författare)
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Swaminathan, Kunchithapadam (författare)
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Patel, Bharat K. C. (författare)
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- Divne, Christina (författare)
- KTH,Glykovetenskap
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(creator_code:org_t)
- Elsevier BV, 2008
- 2008
- Engelska.
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 378:4, s. 852-870
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Nyckelord
- 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
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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