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PDBsum entry 1wmb
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Oxidoreductase
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PDB id
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1wmb
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References listed in PDB file
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Key reference
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Title
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D-3-Hydroxybutyrate dehydrogenase from pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme.
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Authors
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K.Ito,
Y.Nakajima,
E.Ichihara,
K.Ogawa,
N.Katayama,
K.Nakashima,
T.Yoshimoto.
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Ref.
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J Mol Biol, 2006,
355,
722-733.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 4.
Figure 4. The stereo diagram of SDRs superimposed onto
ligand-free HBDH. The C^a trace of HBDH is shown by the orange
line, and 2hsd, 1ae1, 1k2w, 1gco, 1hxh, and 1ahh by red, blue,
cyan, blue-violet, magenta, and green lines, respectively.
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Figure 5.
Figure 5. Drawing of the NAD^+ binding site in HBDH. (a)
The wire frame shows the F[o] -F[c] omit map for NAD^+ and
cacodylate anion contoured at 2s levels. The stick models show
residues interacting with NAD^+ or cacodylate anion. (b) The
scheme diagram shows hydrogen-bonding interaction with NAD^+.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
355,
722-733)
copyright 2006.
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