title: Molecular basis of substrate recognition in D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida.
abstract: D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida (EC 1.1.1.30) belongs to the family of short-chain dehydrogenases/reductases (SDRs). It catalyzes the reversible and stereospecific oxidation of D-3-hydroxybutyrate (D-3-HB) to acetoacetate with the aid of NAD(+) as coenzyme. This study contributes to understanding the mechanism and the high specificity of this enzyme towards its negatively charged and hydrophilic substrate. Sequence comparison of 44 bacterial HBDHs shows the residues Gln91, His141, Lys149, Lys192, and Gln193 to be strictly conserved. Site-directed mutagenesis of these amino acids to alanine and subsequent kinetic characterization of the mutated enzymes provides insight into the importance of these residues for substrate recognition and catalysis. Docking studies and molecular-dynamics simulations based on a three-dimensional structure model of a complex between P. putida HBDH and its coenzyme obtained by comparative molecular modeling were performed and provided deeper insight into the binding of the ligands at the molecular level. They show the residues Gln91, His141, Gln193, and, in particular, Lys149 to be involved in a hydrogen-bonding network with the carboxylate group of the substrate.
authors.affiliation: Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology, University of Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Feller
authors.forename: Robert
authors.initials: R
authors.lastname: Günther
authors.forename: Hans-Jörg
authors.initials: HJ
authors.lastname: Hofmann
authors.forename: Marlis
authors.initials: M
authors.lastname: Grunow
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D019344:Lactic Acid / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009243:NAD; D019344:Lactic Acid; D006884:Hydroxybutyrate Dehydrogenase; D020155:3-Hydroxybutyric Acid