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Journal of Biochemistry Advance Access originally published online on January 3, 2009
Journal of Biochemistry 2009 145(4):467-479; doi:10.1093/jb/mvn186
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

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

Kanako Nakashima, Kiyoshi Ito*, Yoshitaka Nakajima, Ryuji Yamazawa, Syunsuke Miyakawa and Tadashi Yoshimoto

Department of Molecular Medicinal Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan

*To whom correspondence should be addressed. Tel: +81-95-819-2436, Fax: +81-95-819-2478, E-mail: k-ito{at}nagasaki-u.ac.jp

Received November 30, 2008; Accepted December 23, 2008


  Abstract

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 Ki value comparable to the Km 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.

Key Words: conformational change, crystal structure, hydroxybutyrate dehydrogenase, short-chain dehydrogenase/reductase family, substrate recognition

Abbreviations: 3-HB, 3-hydroxybutyrate; HBDH, D-3-hydroxybutyrate dehydrogenase; HSDH, hydroxysteroid dehydrogenase; rms, root mean square; SDR, short-chain dehydrogenase/reductase


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