1x1t

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(New page: 200px<br /><applet load="1x1t" size="450" color="white" frame="true" align="right" spinBox="true" caption="1x1t, resolution 1.52&Aring;" /> '''Crystal Structure of...)
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'''Crystal Structure of D-3-Hydroxybutyrate Dehydrogenase from Pseudomonas fragi Complexed with NAD+'''<br />
'''Crystal Structure of D-3-Hydroxybutyrate Dehydrogenase from Pseudomonas fragi Complexed with NAD+'''<br />
==Overview==
==Overview==
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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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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==
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1X1T 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, CAC and NAD 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://ispc.weizmann.ac.il/oca-bin/ocashort?id=1X1T OCA].
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1X1T 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>, <scene name='pdbligand=CAC:'>CAC</scene> and <scene name='pdbligand=NAD:'>NAD</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=1X1T OCA].
==Reference==
==Reference==
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[[Category: short chain dehydrogenase]]
[[Category: short chain dehydrogenase]]
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''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Sun Nov 25 01:17:10 2007''
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:50:24 2008''

Revision as of 13:50, 21 February 2008

Image:1x1t.gif

1x1t, resolution 1.52Å

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

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

1X1T 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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