Abstract
Meso-2,3-butanediol dehydrogenase (meso-2,3-BDH) catalyzes NAD+ -dependent conversion of meso-2,3-butanediol to acetoin, a crucial external energy storage molecule in fermentive bacteria. In this study, the active tunnel of meso-2,3-BDH was identified. The two short α helixes positioned away from the α4-helix possibly expose the hydrophobic ligand-binding cavity, gating the exit of product and cofactor from the activity pocket. Further MM/GBSA-binding free energy analysis shows that Phe212 and Asn146 function as the key product-release sites. Site-directed mutagenesis experiments targeted to the sites show that the kcat of Phe212Tyr is enhanced up to (4-8)-fold. The original activity of Asn146Gln is retained, but the activity of Asn146Ala mutation is lost. These results could provide helpful guidance on rational design of short-chain dehydrogenases/reductases.
Keywords:
meso-2,3-butanediol dehydrogenase; rational design; short-chain dehydrogenase/reductase.
© 2017 Federation of European Biochemical Societies.
MeSH terms
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Acetoin / chemistry
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Acetoin / metabolism
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Alcohol Oxidoreductases / chemistry*
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Alcohol Oxidoreductases / genetics
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Alcohol Oxidoreductases / metabolism
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Amino Acid Sequence
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Bacterial Proteins / chemistry*
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Binding Sites
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Butylene Glycols / chemistry
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Butylene Glycols / metabolism
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Cloning, Molecular
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Crystallography, X-Ray
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Gene Expression
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Kinetics
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Klebsiella pneumoniae / enzymology
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Klebsiella pneumoniae / genetics*
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Models, Molecular
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Molecular Docking Simulation*
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Molecular Dynamics Simulation*
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Mutagenesis, Site-Directed
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Protein Binding
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Protein Conformation, alpha-Helical
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Protein Engineering
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Protein Interaction Domains and Motifs
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Sequence Alignment
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Sequence Homology, Amino Acid
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Substrate Specificity
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Thermodynamics
Substances
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Bacterial Proteins
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Butylene Glycols
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Recombinant Proteins
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2,3-butylene glycol
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Acetoin
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Alcohol Oxidoreductases
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butanediol dehydrogenase