1X1T

Crystal Structure of D-3-Hydroxybutyrate Dehydrogenase from Pseudomonas fragi Complexed with NAD+

Summary for 1X1T

• Entry DOI: 10.2210/pdb1x1t/pdb
• Related: 1WMB
• Descriptor: D(-)-3-hydroxybutyrate dehydrogenase, MAGNESIUM ION, CACODYLATE ION, ... (5 entities in total)
• Functional Keywords: nad, nadh, sdr, short chain dehydrogenase, ketone body, beta-hydroxybutyrate, oxidoreductase
• Biological source: Pseudomonas fragi
• Total number of polymer chains: 1
• Total formula weight: 27537.13

Authors

Ito, K.,Nakajima, Y.,Ichihara, E.,Ogawa, K.,Yoshimoto, T. (deposition date: 2005-04-13, release date: 2006-01-10, Last modification date: 2023-10-25)

Primary citation

Ito, K.,Nakajima, Y.,Ichihara, E.,Ogawa, K.,Katayama, N.,Nakashima, K.,Yoshimoto, T.
d-3-Hydroxybutyrate Dehydrogenase from Pseudomonas fragi: Molecular Cloning of the Enzyme Gene and Crystal Structure of the Enzyme
J.Mol.Biol., 355:722-733, 2006

PubMed 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.

PubMed: 16325199
DOI: 10.1016/j.jmb.2005.10.072

Experimental method

X-RAY DIFFRACTION (1.52 Å)