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 degrees . However, the two small alpha-helices (alphaFG1 and alphaFG2) 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.
Paithankar
Karthik S
KS
Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Leipzig, Germany.
Feller
Claudia
C
Kuettner
E Bartholomeus
EB
Keim
Antje
A
Grunow
Marlis
M
Sträter
Norbert
N
D020155:3-Hydroxybutyric Acid / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D044206:Pseudomonas fragi / Q000201:enzymology; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D009243:NAD; D006884:Hydroxybutyrate Dehydrogenase; D020155:3-Hydroxybutyric Acid