D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme.
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.
Ito
Kiyoshi
K
Department of Molecular Medicinal Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. k.ito@net.nagasaki-u.ac.jp
Nakajima
Yoshitaka
Y
Ichihara
Emi
E
Ogawa
Kyohei
K
Katayama
Naoko
N
Nakashima
Kanako
K
Yoshimoto
Tadashi
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D002101:Cacodylic Acid / Q000737:chemistry; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044206:Pseudomonas fragi / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D008024:Ligands; D021122:Protein Subunits; D009243:NAD; D002101:Cacodylic Acid; D006884:Hydroxybutyrate Dehydrogenase