Abstract
A beta-xylosidase from Bacillus stearothermophilus T-6 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan. The conserved Glu160 was proposed as the acid-base catalyst. An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid-base catalytic residue.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Azides / pharmacology
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Binding Sites / drug effects
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Binding Sites / physiology
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Catalysis
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Cloning, Molecular
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Dose-Response Relationship, Drug
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Escherichia coli / genetics
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Geobacillus stearothermophilus / enzymology*
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Glutamic Acid / genetics
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Glutamic Acid / metabolism*
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Glycosides / metabolism
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Hydrolysis / drug effects
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Molecular Sequence Data
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Sequence Analysis, DNA
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Sequence Homology, Amino Acid
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Substrate Specificity / physiology
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Xylosidases / genetics*
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Xylosidases / metabolism*
Substances
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Azides
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Glycosides
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4-nitrophenyl beta-D-xyloside
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Glutamic Acid
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Xylosidases
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exo-1,4-beta-D-xylosidase