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

Abstract

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.

Structural and biochemical characterization of an atypical short‐chain dehydrogenase/reductase reveals an unusual cofactor preference

“…As seen for other NADPH co-complexes at the same resolution, the nicotinamide ring of the cofactor adopts the nonplanar, nonaromatic state consistent with the reduced form. Interestingly, helix a 8 and its associated N-and C-terminal loops (residues 163-185), a region harbouring the substrate binding motif in catalytically competent SDRs, exhibited flexibility in two of the four subunits, as demonstrated by higher B-factors on average, which is consistent with the behaviour of this structural element in canonical SDR [14,26] (Fig. 4B).…”

The structures ofAlcaligenes faecalisD-3-hydroxybutyrate dehydrogenase before and after NAD+and acetate binding suggest a dynamical reaction mechanism as a member of the SDR family

“…An alternative methyl C atom with the S configuration is difficult to accommodate in the enzyme.3 The substrate-bound model proposed byPaithankar et al (2007) is absolutely inconsistent with our observed structure.…”

“…1 In the apoenzyme, the temperature factors of these residues are relatively high with a wide range in the eight subunits depending on the crystal packing. Such flexibility of the domain has been exemplified in the crystal structure of Pseudomonas putida HBDH (Paithankar et al, 2007), which was published just before the submission of our manuscript.…”

Closed Complex of the D-3-Hydroxybutyrate Dehydrogenase Induced by an Enantiomeric Competitive Inhibitor

“…DHRS6 is a human cytosolic type II HBDH having high K m and low k cat values (12). Two bacterial HBDHs, one from Pseudomonas putida (PpHBDH) and the other from Alcaligenes faecalis (AfHBDH), were also reported (13,14). The structures of bacterial HBDHs contained a closed conformation.…”