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.

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Short-chain dehydrogenases ⁄ reductases (SDRs) constitute a large protein family that now includes more than 1000 enzymes in humans, mammals, insects and bacteria [1]. The dehydrogenases act on a wide variety of substrates, including steroids, retinoids, prostaglandins, sugars and alcohols. The name SDR is based on their smaller subunit size of about 250 residues compared with the medium-chain dehydrogenase ⁄ reductase family that has a subunit size of about 350 residues. The SDR enzymes exhibit a sequence identity of 15-30% and share two signature motifs: a GxxxGxG motif involved in coenzyme binding; and a YxxxK D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases ⁄ reductases. We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 Å . The socalled substrate-binding loop (residues 187-210) was partially disordered in several subunits, in both the presence and absence of NAD + . However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD + . Structural comparisons indicated that the loop moves as a rigid body by about 46°. However, the two small a-helices (aFG1 and aFG2) of the loop also re-orientated slightly during the conformational change. Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change. A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD + binding, is positioned competent for catalysis. Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate. A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal. NAD + binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form. The finding that all NAD + -bound subunits are present in the closed form and all NAD + -free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone. This mechanism may contribute to the sequential binding of cosubstrate followed by substrate.Abbreviations PfHBDH, Pseudomonas fragi D-3-hydroxybutyrate dehydrogenase; PpHBDH, Pseudomonas putida D-3-hydroxybutyrate dehydrogenase; SDR, short-chain dehydrogenase ⁄ reductase.

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