1wmb
From Proteopedia
(New page: 200px<br /><applet load="1wmb" size="450" color="white" frame="true" align="right" spinBox="true" caption="1wmb, resolution 2.0Å" /> '''Crystal structure of ...) |
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| - | [[Image:1wmb.gif|left|200px]]<br /><applet load="1wmb" size=" | + | [[Image:1wmb.gif|left|200px]]<br /><applet load="1wmb" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1wmb, resolution 2.0Å" /> | caption="1wmb, resolution 2.0Å" /> | ||
'''Crystal structure of NAD dependent D-3-hydroxybutylate dehydrogenase'''<br /> | '''Crystal structure of NAD dependent D-3-hydroxybutylate dehydrogenase'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The gene coding for d-3-hydroxybutyrate dehydrogenase (HBDH) was cloned | + | 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. |
==About this Structure== | ==About this Structure== | ||
| - | 1WMB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_fragi Pseudomonas fragi] with MG and CAC as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/3-hydroxybutyrate_dehydrogenase 3-hydroxybutyrate dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.30 1.1.1.30] Full crystallographic information is available from [http:// | + | 1WMB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_fragi Pseudomonas fragi] with <scene name='pdbligand=MG:'>MG</scene> and <scene name='pdbligand=CAC:'>CAC</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/3-hydroxybutyrate_dehydrogenase 3-hydroxybutyrate dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.30 1.1.1.30] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1WMB OCA]. |
==Reference== | ==Reference== | ||
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[[Category: short chain dehydrogenase]] | [[Category: short chain dehydrogenase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:45:58 2008'' |
Revision as of 13:46, 21 February 2008
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Crystal structure of NAD dependent D-3-hydroxybutylate dehydrogenase
Overview
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.
About this Structure
1WMB is a Single protein structure of sequence from Pseudomonas fragi with and as ligands. Active as 3-hydroxybutyrate dehydrogenase, with EC number 1.1.1.30 Full crystallographic information is available from OCA.
Reference
D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme., Ito K, Nakajima Y, Ichihara E, Ogawa K, Katayama N, Nakashima K, Yoshimoto T, J Mol Biol. 2006 Jan 27;355(4):722-33. Epub 2005 Nov 14. PMID:16325199
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