Structural and catalytic properties of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas aeruginosa.
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.
Mountassif
Driss
D
INSERM U866; Université de Bourgogne, LBMC (Biochimie Métabolique et Nutritionnelle), Faculté des Sciences, 6 Bd Gabriel, 21000, Dijon cedex, France.
Andreoletti
Pierre
P
Cherkaoui-Malki
Mustapha
M
Latruffe
Norbert
N
El Kebbaj
M'hammed Saïd
MS
D020155:3-Hydroxybutyric Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D055162:Biocatalysis; D002846:Chromatography, Affinity; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D012685:Sepharose / Q000031:analogs & derivatives
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C017842:cibacron blue-sepharose; D009243:NAD; D012685:Sepharose; D006884:Hydroxybutyrate Dehydrogenase; D020155:3-Hydroxybutyric Acid