Cosubstrate‐induced dynamics of D‐3‐hydroxybutyrate dehydrogenase from Pseudomonas putida

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 A. The so-called 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 α-helices (αFG1 and αFG2) 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.