1. D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme.
- Author
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Ito K, Nakajima Y, Ichihara E, Ogawa K, Katayama N, Nakashima K, and Yoshimoto T
- Subjects
- Amino Acid Sequence, Binding Sites, Cacodylic Acid chemistry, Cloning, Molecular, Conserved Sequence, Crystallography, X-Ray, Enzyme Inhibitors chemistry, Gene Expression genetics, Humans, Hydroxybutyrate Dehydrogenase antagonists & inhibitors, Hydroxybutyrate Dehydrogenase genetics, Ligands, Molecular Sequence Data, NAD chemistry, NAD metabolism, Protein Structure, Quaternary, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, Pseudomonas fragi genetics, Sequence Alignment, Sequence Homology, Amino Acid, Substrate Specificity, Hydroxybutyrate Dehydrogenase chemistry, Hydroxybutyrate Dehydrogenase metabolism, Pseudomonas fragi enzymology
- Abstract
The gene coding for d-3-hydroxybutyrate dehydrogenase (HBDH) was cloned from Pseudomonas fragi. The nucleotide sequence contained a 780 bp open reading frame encoding a 260 amino acid residue protein. The recombinant enzyme was efficiently expressed in Escherichia coli cells harboring pHBDH11 and was purified to homogeneity as judged by SDS-PAGE. The enzyme showed a strict stereospecificity to the D-enantiomer (3R-configuration) of 3-hydroxybutyrate as a substrate. Crystals of the ligand-free HBDH and of the enzyme-NAD+ complex were obtained using the hanging-drop, vapor-diffusion method. The crystal structure of the HBDH was solved by the multiwavelength anomalous diffraction method using the SeMet-substituted enzyme and was refined to 2.0 A resolution. The overall structure of P.fragi HBDH, including the catalytic tetrad of Asn114, Ser142, Tyr155, and Lys159, shows obvious relationships with other members of the short-chain dehydrogenase/reductase (SDR) family. A cacodylate anion was observed in both the ligand-free enzyme and the enzyme-NAD+ complex, and was located near the catalytic tetrad. It was shown that the cacodylate inhibited the NAD+-dependent D-3-hydroxybutyrate dehydrogenation competitively, with a Ki value of 5.6 mM. From the interactions between cacodylate and the enzyme, it is predicted that substrate specificity is achieved through the recognition of the 3-methyl and carboxyl groups of the substrate.
- Published
- 2006
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