2phh: Difference between revisions

Revision as of 19:29, 21 February 2008

THE COENZYME ANALOGUE ADENOSINE 5-DIPHOSPHORIBOSE DISPLACES FAD IN THE ACTIVE SITE OF P-HYDROXYBENZOATE HYDROXYLASE. AN X-RAY CRYSTALLOGRAPHIC INVESTIGATION

OverviewOverview

p-Hydroxybenzoate hydroxylase (PHBH) is an NADPH-dependent enzyme. To locate the NADPH binding site, the enzyme was crystallized under anaerobic conditions in the presence of the substrate p-hydroxybenzoate, the coenzyme analogue adenosine 5-diphosphoribose (ADPR), and sodium dithionite. This yielded colorless crystals that were suitable for X-ray analysis. Diffraction data were collected up to 2.7-A resolution. A difference Fourier between data from these colorless crystals and data from yellow crystals of the enzyme-substrate complex showed that in the colorless crystals the flavin ring was absent. The adenosine 5'-diphosphate moiety, which is the common part between FAD and ADPR, was still present. After restrained least-squares refinement of the enzyme-substrate complex with the riboflavin omitted from the model, additional electron density appeared near the pyrophosphate, which indicated the presence of an ADPR molecule in the FAD binding site of PHBH. The complete ADPR molecule was fitted to the electron density, and subsequent least-squares refinement resulted in a final R factor of 16.8%. Replacement of bound FAD by ADPR was confirmed by equilibrium dialysis, where it was shown that ADPR can effectively remove FAD from the enzyme under mild conditions in 0.1 M potassium phosphate buffer, pH 8.0. The empty pocket left by the flavin ring is filled by solvent, leaving the architecture of the active site and the binding of the substrate largely unaffected.

About this StructureAbout this Structure

2PHH is a Single protein structure of sequence from Pseudomonas fluorescens with and as ligands. Active as 4-hydroxybenzoate 3-monooxygenase, with EC number 1.14.13.2 Full crystallographic information is available from OCA.

ReferenceReference

The coenzyme analogue adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation., van der Laan JM, Schreuder HA, Swarte MB, Wierenga RK, Kalk KH, Hol WG, Drenth J, Biochemistry. 1989 Sep 5;28(18):7199-205. PMID:2819062

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-[[Image:2phh.gif|left|200px]]<br /><applet load="2phh" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:2phh.gif|left|200px]]<br /><applet load="2phh" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="2phh, resolution 2.7&Aring;" />
 '''THE COENZYME ANALOGUE ADENOSINE 5-DIPHOSPHORIBOSE DISPLACES FAD IN THE ACTIVE SITE OF P-HYDROXYBENZOATE HYDROXYLASE. AN X-RAY CRYSTALLOGRAPHIC INVESTIGATION'''<br />
 ==Overview==
-p-Hydroxybenzoate hydroxylase (PHBH) is an NADPH-dependent enzyme. To, locate the NADPH binding site, the enzyme was crystallized under anaerobic, conditions in the presence of the substrate p-hydroxybenzoate, the, coenzyme analogue adenosine 5-diphosphoribose (ADPR), and sodium, dithionite. This yielded colorless crystals that were suitable for X-ray, analysis. Diffraction data were collected up to 2.7-A resolution. A, difference Fourier between data from these colorless crystals and data, from yellow crystals of the enzyme-substrate complex showed that in the, colorless crystals the flavin ring was absent. The adenosine, 5'-diphosphate moiety, which is the common part between FAD and ADPR, was, still present. After restrained least-squares refinement of the, enzyme-substrate complex with the riboflavin omitted from the model, additional electron density appeared near the pyrophosphate, which, indicated the presence of an ADPR molecule in the FAD binding site of, PHBH. The complete ADPR molecule was fitted to the electron density, and, subsequent least-squares refinement resulted in a final R factor of 16.8%., Replacement of bound FAD by ADPR was confirmed by equilibrium dialysis, where it was shown that ADPR can effectively remove FAD from the enzyme, under mild conditions in 0.1 M potassium phosphate buffer, pH 8.0. The, empty pocket left by the flavin ring is filled by solvent, leaving the, architecture of the active site and the binding of the substrate largely, unaffected.
+p-Hydroxybenzoate hydroxylase (PHBH) is an NADPH-dependent enzyme. To locate the NADPH binding site, the enzyme was crystallized under anaerobic conditions in the presence of the substrate p-hydroxybenzoate, the coenzyme analogue adenosine 5-diphosphoribose (ADPR), and sodium dithionite. This yielded colorless crystals that were suitable for X-ray analysis. Diffraction data were collected up to 2.7-A resolution. A difference Fourier between data from these colorless crystals and data from yellow crystals of the enzyme-substrate complex showed that in the colorless crystals the flavin ring was absent. The adenosine 5'-diphosphate moiety, which is the common part between FAD and ADPR, was still present. After restrained least-squares refinement of the enzyme-substrate complex with the riboflavin omitted from the model, additional electron density appeared near the pyrophosphate, which indicated the presence of an ADPR molecule in the FAD binding site of PHBH. The complete ADPR molecule was fitted to the electron density, and subsequent least-squares refinement resulted in a final R factor of 16.8%. Replacement of bound FAD by ADPR was confirmed by equilibrium dialysis, where it was shown that ADPR can effectively remove FAD from the enzyme under mild conditions in 0.1 M potassium phosphate buffer, pH 8.0. The empty pocket left by the flavin ring is filled by solvent, leaving the architecture of the active site and the binding of the substrate largely unaffected.
 ==About this Structure==
-PHH is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_fluorescens Pseudomonas fluorescens] with APR and PHB as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/4-hydroxybenzoate_3-monooxygenase 4-hydroxybenzoate 3-monooxygenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.14.13.2 1.14.13.2] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2PHH OCA].
+PHH is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_fluorescens Pseudomonas fluorescens] with <scene name='pdbligand=APR:'>APR</scene> and <scene name='pdbligand=PHB:'>PHB</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/4-hydroxybenzoate_3-monooxygenase 4-hydroxybenzoate 3-monooxygenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.14.13.2 1.14.13.2] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2PHH OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Pseudomonas fluorescens]]
 [[Category: Single protein]]
-[[Category: Derlaan, J.M.Van.]]
+[[Category: Derlaan, J M.Van.]]
 [[Category: Drenth, J.]]
-[[Category: Hol, W.G.J.]]
+[[Category: Hol, W G.J.]]
 [[Category: APR]]
 [[Category: PHB]]
 [[Category: oxidoreductase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 13:31:58 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 18:29:26 2008''