Molecular Basis of Substrate Recognition in D‐3‐Hydroxybutyrate Dehydrogenase from Pseudomonas putida

Abstract

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.

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

“…A model for the binding mode of the substrate D‐3‐hydroxybutyrate to HBDH has been suggested based on a homology model of Pp HBDH and molecular modelling techniques [9]. In this model, the side chain of Gln193 belonging to the substrate‐binding loop forms hydrogen bonds to the carboxylate group of the substrate.…”

“…Further polar interactions to the substrate are formed by Lys149, Gln91, His141 and Tyr152. Of these residues, the important function for substrate binding has been demonstrated for a Gln91Ala mutant with K m 51 m m and k cat 411·s −1 and for a Lys149Ala mutant that was essentially inactive [9]. His141 appears to be also important for efficient catalysis because in a His141Ala mutant the K m increased to only 4 m m , whereas the k cat decreased significantly to 13·s −1 .…”

“…D‐3‐Hydroxybutyrate dehydrogenase from Pseudomonas putida ( Pp HBDH) (EC 1.1.1.30, GenBank accession number .2) catalyzes the reversible and stereospecific oxidation of D‐3‐hydroxybutyrate to acetoacetate using NAD + as a coenzyme. One subunit contains 256 amino acids with a calculated molecular weight of ≈ 26.6 kDa [9]. X‐ray structures of the homologous enzyme from Pseudomonas fragi ( Pf HBDH) in the presence of NAD + and inhibitor (Protein Data Bank accession code: 1X1T) and without cosubstrate (Protein Data Bank accession code: 1WMB) have been recently determined [10].…”

“…In addition, a crystal structure of a human cytosolic HBDH (DHRS6) with bound NAD + is available [11]. Based on homology modelling, substrate and inhibitor docking studies, and site‐directed mutagenesis, residues Gln91, His141, Lys149, Tyr152 and Gln193 were found to be involved in substrate binding in Pp HBDH [9]. With the exception of Gln193, these residues are located in the deep active‐site cleft.…”

The metabolism of (R)-3-hydroxybutyrate is regulated by the enhancer-binding protein PA2005 and the alternative sigma factor RpoN in Pseudomonas aeruginosa PAO1

“…Nonetheless, these bacteria possess an NAD + -dependent dehydrogenase (BdhA) that converts (R)-3-HB into acetoacetate, thereby allowing these bacteria to use (R)-3-HB as a growth substrate (Feller et al, 2006;Ito et al, 2006;Mountassif et al, 2010). BdhA dehydrogenases have been biochemically characterized for some species of Pseudomonas, including P. putida (Feller et al, 2006;Paithankar et al, 2007) …”