Abstract
To put forward BDH from Pseudomonas aeruginosa's enzymatic properties, we report a two-step purification of BDH and its gene sequencing allowing the investigation of its structural properties. Purification of BDH was achieved, using ammonium sulfate fractionation and Blue Sepharose CL-6B affinity chromatography. SDS-PAGE analysis reveals a MM of 29 kDa, whereas the native enzyme showed a MM of 120 kDa suggesting a homotetrameric structure. BDH encoding gene sequence shows a nucleotide open reading frame sequence of 771 bp encoding a 265 amino acid residues polypeptide chain. The modeling analysis of the three dimensional structure fits with the importance of amino acids in the catalysis reaction especially a strictly conserved tetrad. Amino-acid residues in interaction with the coenzyme NAD(+) were also identified.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3-Hydroxybutyric Acid / 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 / isolation & purification
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Bacterial Proteins / metabolism
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Biocatalysis
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Chromatography, Affinity
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DNA, Bacterial / chemistry
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DNA, Bacterial / genetics
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Electrophoresis, Polyacrylamide Gel
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Hydroxybutyrate Dehydrogenase / chemistry*
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Hydroxybutyrate Dehydrogenase / genetics
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Hydroxybutyrate Dehydrogenase / isolation & purification
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Hydroxybutyrate Dehydrogenase / metabolism*
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Models, Molecular
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Molecular Sequence Data
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Molecular Weight
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NAD / metabolism
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Protein Conformation
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Pseudomonas aeruginosa / enzymology*
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Pseudomonas aeruginosa / genetics
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Sepharose / analogs & derivatives
Substances
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Bacterial Proteins
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DNA, Bacterial
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cibacron blue-sepharose
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NAD
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Sepharose
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Hydroxybutyrate Dehydrogenase
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3-Hydroxybutyric Acid