Closed complex of the D-3-hydroxybutyrate dehydrogenase induced by an enantiomeric competitive inhibitor.
D-3-Hydroxybutyrate dehydrogenase (HBDH) from Pseudomonas fragi showed a strict stereospecificity to the d-enantiomer of 3-hydroxybutyrate (d-3-HB) as a substrate. The l-enantiomer acts as a competitive inhibitor, with a K(i) value comparable to the K(m) value for d-3-HB. We have determined the crystal structures of the ternary complex of HBDH-NAD(+)-l-3-HB and the binary complex of HBDH-NAD(+). The former structure showed a so-called closed-form conformation, which is considered an active form for catalysis, while the latter stayed mostly in a open-form conformation. The determined structures along with the site-directed mutagenesis confirmed the substrate recognition mechanism that we proposed previously. The hydrogen bonding interaction between Gln196, located in the moving helix, and the carboxyl group of the substrate/inhibitor is important for the stable ternary complex formation. Finally, the crystal structures of the Thr190 mutants, T190S and T190A, indicate that the Thr190 is a key residue for the open-closed conformational change. T190S retained 37% of the activity. In T190A, however, the activity decreased to 0.1% that of the wild-type enzyme. Fixing the position of the hydroxyl group of Thr190 to form hydrogen bonds to the pyrophosphate moiety and the carboxamide of NAD(+) seems to be a significant factor for the open-closed conformational change.
Nakashima
Kanako
K
Department of Molecular Medicinal Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Ito
Kiyoshi
K
Nakajima
Yoshitaka
Y
Yamazawa
Ryuji
R
Miyakawa
Syunsuke
S
Yoshimoto
Tadashi
T
D020155:3-Hydroxybutyric Acid / Q000737:chemistry* / Q000494:pharmacology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry; D044206:Pseudomonas fragi / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000187:drug effects
D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D021122:Protein Subunits; D006884:Hydroxybutyrate Dehydrogenase; D020155:3-Hydroxybutyric Acid