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Cosubstrate‐induced dynamics of D‐3‐hydroxybutyrate dehydrogenase from Pseudomonas putida
Author(s) -
Paithankar Karthik S.,
Feller Claudia,
Kuettner E. Bartholomeus,
Keim Antje,
Grunow Marlis,
Sträter Norbert
Publication year - 2007
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2007.06102.x
Subject(s) - pseudomonas putida , nad+ kinase , cofactor , stereochemistry , conformational change , chemistry , substrate (aquarium) , dehydrogenase , crystallography , binding site , biophysics , enzyme , biochemistry , biology , ecology
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 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.