2b7r: Difference between revisions

Revision as of 17:35, 21 February 2008

Structure of E378D mutant flavocytochrome c3

OverviewOverview

The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their k(cat) values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.

About this StructureAbout this Structure

2B7R is a Single protein structure of sequence from Shewanella frigidimarina with , , and as ligands. Active as Succinate dehydrogenase, with EC number 1.3.99.1 Full crystallographic information is available from OCA.

ReferenceReference

A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina., Pankhurst KL, Mowat CG, Rothery EL, Hudson JM, Jones AK, Miles CS, Walkinshaw MD, Armstrong FA, Reid GA, Chapman SK, J Biol Chem. 2006 Jul 21;281(29):20589-97. Epub 2006 May 12. PMID:16699170

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-[[Image:2b7r.gif|left|200px]]<br /><applet load="2b7r" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:2b7r.gif|left|200px]]<br /><applet load="2b7r" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="2b7r, resolution 1.70&Aring;" />
 '''Structure of E378D mutant flavocytochrome c3'''<br />
 ==Overview==
-The mechanism for fumarate reduction by the soluble fumarate reductase, from Shewanella frigidimarina involves hydride transfer from FAD and, proton transfer from the active-site acid, Arg-402. It has been proposed, that Arg-402 forms part of a proton transfer pathway that also involves, Glu-378 and Arg-381 but, unusually, does not involve any bound water, molecules. To gain further insight into the importance of this proton, pathway we have perturbed it by substituting Arg-381 by lysine and, methionine and Glu-378 by aspartate. Although all the mutant enzymes, retain measurable activities, there are orders-of-magnitude decreases in, their k(cat) values compared with the wild-type enzyme. Solvent kinetic, isotope effects show that proton transfer is rate-limiting in the, wild-type and mutant enzymes. Proton inventories indicate that the proton, pathway involves multiple exchangeable groups. Fast scan protein-film, voltammetric studies on wild-type and R381K enzymes show that the proton, transfer pathway delivers one proton per catalytic cycle and is not, required for transporting the other proton, which transfers as a hydride, from the reduced, protonated FAD. The crystal structures of E378D and, R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that, disturb proton transport. Taken together, the results indicate that, Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely, conserved throughout the fumarate reductase/succinate dehydrogenase family, of enzymes.
+The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their k(cat) values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.
 ==About this Structure==
-B7R is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Shewanella_frigidimarina Shewanella frigidimarina] with NA, HEM, FAD and FUM as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Succinate_dehydrogenase Succinate dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.3.99.1 1.3.99.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2B7R OCA].
+B7R is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Shewanella_frigidimarina Shewanella frigidimarina] with <scene name='pdbligand=NA:'>NA</scene>, <scene name='pdbligand=HEM:'>HEM</scene>, <scene name='pdbligand=FAD:'>FAD</scene> and <scene name='pdbligand=FUM:'>FUM</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Succinate_dehydrogenase Succinate dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.3.99.1 1.3.99.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2B7R OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Single protein]]
 [[Category: Succinate dehydrogenase]]
-[[Category: Chapman, S.K.]]
+[[Category: Chapman, S K.]]
-[[Category: Miles, C.S.]]
+[[Category: Miles, C S.]]
-[[Category: Mowat, C.G.]]
+[[Category: Mowat, C G.]]
-[[Category: Pankhurst, K.L.]]
+[[Category: Pankhurst, K L.]]
-[[Category: Reid, G.A.]]
+[[Category: Reid, G A.]]
-[[Category: Rothery, E.L.]]
+[[Category: Rothery, E L.]]
-[[Category: Walkinshaw, M.D.]]
+[[Category: Walkinshaw, M D.]]
 [[Category: FAD]]
 [[Category: FUM]]
@@ Line 29: / Line 29: @@
 [[Category: proton delivery]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 08:37:39 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:35:00 2008''