2jbv
From Proteopedia
(New page: 200px<br /><applet load="2jbv" size="350" color="white" frame="true" align="right" spinBox="true" caption="2jbv, resolution 1.86Å" /> '''CRYSTAL STRUCTURE OF...) |
|||
| Line 4: | Line 4: | ||
==Overview== | ==Overview== | ||
| - | Choline oxidase catalyzes the oxidation of choline to glycine betaine, a | + | Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Nepsilon2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 value similar to that of the wild-type enzyme but kcat and kcat/Km values that were 230 and 35 times lower, respectively, than in the wild-type enzyme. These data are consistent with the spatial location of the negative charge on residue 312 being important for the oxidation of the alcohol substrate. Solvent viscosity and substrate kinetic isotope effects suggest the presence of an internal equilibrium in the Glu312Asp enzyme prior to the hydride transfer reaction. Altogether, the crystallographic and mechanistic data suggest that Glu312 is important for binding and positioning of the substrate in the active site of choline oxidase. |
==About this Structure== | ==About this Structure== | ||
| - | 2JBV is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Arthrobacter_globiformis Arthrobacter globiformis] with <scene name='pdbligand=FAO:'>FAO</scene>, <scene name='pdbligand=DMS:'>DMS</scene> and <scene name='pdbligand=UNX:'>UNX</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Oxidoreductase Oxidoreductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.3.17 1.1.3.17] Known structural/functional Sites: <scene name='pdbsite=AC1:Fao Binding Site For Chain A'>AC1</scene>, <scene name='pdbsite=AC2:Dms Binding Site For Chain A'>AC2</scene>, <scene name='pdbsite=AC3:Fao Binding Site For Chain B'>AC3</scene>, <scene name='pdbsite=AC4:Dms Binding Site For Chain B'>AC4</scene>, <scene name='pdbsite=AC5:Unx Binding Site For Chain A'>AC5</scene>, <scene name='pdbsite=AC6:Unx Binding Site For Chain A'>AC6</scene>, <scene name='pdbsite=AC7:Unx Binding Site For Chain B'>AC7</scene> and <scene name='pdbsite=AC8:Unx Binding Site For Chain B'>AC8</scene>. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2JBV OCA]. | + | 2JBV is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Arthrobacter_globiformis Arthrobacter globiformis] with <scene name='pdbligand=FAO:'>FAO</scene>, <scene name='pdbligand=DMS:'>DMS</scene> and <scene name='pdbligand=UNX:'>UNX</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Oxidoreductase Oxidoreductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.3.17 1.1.3.17] Known structural/functional Sites: <scene name='pdbsite=AC1:Fao+Binding+Site+For+Chain+A'>AC1</scene>, <scene name='pdbsite=AC2:Dms+Binding+Site+For+Chain+A'>AC2</scene>, <scene name='pdbsite=AC3:Fao+Binding+Site+For+Chain+B'>AC3</scene>, <scene name='pdbsite=AC4:Dms+Binding+Site+For+Chain+B'>AC4</scene>, <scene name='pdbsite=AC5:Unx+Binding+Site+For+Chain+A'>AC5</scene>, <scene name='pdbsite=AC6:Unx+Binding+Site+For+Chain+A'>AC6</scene>, <scene name='pdbsite=AC7:Unx+Binding+Site+For+Chain+B'>AC7</scene> and <scene name='pdbsite=AC8:Unx+Binding+Site+For+Chain+B'>AC8</scene>. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2JBV OCA]. |
==Reference== | ==Reference== | ||
| - | Role of Glu312 in | + | Role of Glu312 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase., Quaye O, Lountos GT, Fan F, Orville AM, Gadda G, Biochemistry. 2008 Jan 8;47(1):243-56. Epub 2007 Dec 12. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=18072756 18072756] |
[[Category: Arthrobacter globiformis]] | [[Category: Arthrobacter globiformis]] | ||
[[Category: Oxidoreductase]] | [[Category: Oxidoreductase]] | ||
| Line 16: | Line 16: | ||
[[Category: Fan, F.]] | [[Category: Fan, F.]] | ||
[[Category: Gadda, G.]] | [[Category: Gadda, G.]] | ||
| - | [[Category: Lountos, G | + | [[Category: Lountos, G T.]] |
| - | [[Category: Orville, A | + | [[Category: Orville, A M.]] |
[[Category: DMS]] | [[Category: DMS]] | ||
[[Category: FAO]] | [[Category: FAO]] | ||
| Line 30: | Line 30: | ||
[[Category: oxidoreductase]] | [[Category: oxidoreductase]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 18:01:30 2008'' |
Revision as of 16:01, 21 February 2008
|
CRYSTAL STRUCTURE OF CHOLINE OXIDASE REVEALS INSIGHTS INTO THE CATALYTIC MECHANISM
Overview
Choline oxidase catalyzes the oxidation of choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of choline oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Nepsilon2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 value similar to that of the wild-type enzyme but kcat and kcat/Km values that were 230 and 35 times lower, respectively, than in the wild-type enzyme. These data are consistent with the spatial location of the negative charge on residue 312 being important for the oxidation of the alcohol substrate. Solvent viscosity and substrate kinetic isotope effects suggest the presence of an internal equilibrium in the Glu312Asp enzyme prior to the hydride transfer reaction. Altogether, the crystallographic and mechanistic data suggest that Glu312 is important for binding and positioning of the substrate in the active site of choline oxidase.
About this Structure
2JBV is a Single protein structure of sequence from Arthrobacter globiformis with , and as ligands. Active as Oxidoreductase, with EC number 1.1.3.17 Known structural/functional Sites: , , , , , , and . Full crystallographic information is available from OCA.
Reference
Role of Glu312 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase., Quaye O, Lountos GT, Fan F, Orville AM, Gadda G, Biochemistry. 2008 Jan 8;47(1):243-56. Epub 2007 Dec 12. PMID:18072756
Page seeded by OCA on Thu Feb 21 18:01:30 2008
