1umd: Difference between revisions

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branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8 with 4-methyl-2-oxopentanoate as an intermediate

OverviewOverview

The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the alpha-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the alpha- and beta-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the alpha-carbanion/enamine intermediate (E1(ceim)). Binding of cofactors to E1(apo) induces a disorder-order transition in two loops adjacent to the active site. Furthermore, upon binding of MPA to E1(holo), the loop comprised of Gly121beta-Gln131beta moves close to the active site and interacts with MPA. The carboxylate group of MPA is recognized mainly by Tyr86beta and N4' of ThDP. The hydrophobic moiety of MPA is recognized by Phe66alpha, Tyr95alpha, Met128alpha and His131alpha. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form. These results suggest that E1 undergoes an open-closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA. ES complex models of E1(holo).MOPA and of E1(ceim).lipoamide built from the above structures suggest that His273alpha and His129beta' are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively.

About this StructureAbout this Structure

1UMD is a Protein complex structure of sequences from Thermus thermophilus with , and as ligands. Active as 3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring), with EC number 1.2.4.4 Full crystallographic information is available from OCA.

ReferenceReference

Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography., Nakai T, Nakagawa N, Maoka N, Masui R, Kuramitsu S, Kamiya N, J Mol Biol. 2004 Apr 2;337(4):1011-33. PMID:15033367

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-[[Image:1umd.gif|left|200px]]<br /><applet load="1umd" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1umd.gif|left|200px]]<br /><applet load="1umd" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1umd, resolution 1.90&Aring;" />
 '''branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8 with 4-methyl-2-oxopentanoate as an intermediate'''<br />
 ==Overview==
-The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo, acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate, (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA, concomitant with the formation of the alpha-carbanion/enamine, intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the, 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2, component. In order to elucidate the catalytic mechanism of E1, the alpha-, and beta-subunits of E1 from Thermus thermophilus HB8 have been, co-expressed in Escherichia coli, purified and crystallized as a stable, complex, and the following crystal structures have been analyzed: the, apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with, the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the, alpha-carbanion/enamine intermediate (E1(ceim)). Binding of cofactors to, E1(apo) induces a disorder-order transition in two loops adjacent to the, active site. Furthermore, upon binding of MPA to E1(holo), the loop, comprised of Gly121beta-Gln131beta moves close to the active site and, interacts with MPA. The carboxylate group of MPA is recognized mainly by, Tyr86beta and N4' of ThDP. The hydrophobic moiety of MPA is recognized by, Phe66alpha, Tyr95alpha, Met128alpha and His131alpha. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation, of the protein loop is almost the same as in the substrate-free, (holoenzyme) form. These results suggest that E1 undergoes an open-closed, conformational change upon formation of the ES complex with a BCOA, and, the mobile region participates in the recognition of the carboxylate group, of the BCOA. ES complex models of E1(holo).MOPA and of E1(ceim).lipoamide, built from the above structures suggest that His273alpha and His129beta', are potential proton donors to the carbonyl group of a BCOA and to the, proximal sulfur atom on the lipoamide, respectively.
+The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the alpha-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the alpha- and beta-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the alpha-carbanion/enamine intermediate (E1(ceim)). Binding of cofactors to E1(apo) induces a disorder-order transition in two loops adjacent to the active site. Furthermore, upon binding of MPA to E1(holo), the loop comprised of Gly121beta-Gln131beta moves close to the active site and interacts with MPA. The carboxylate group of MPA is recognized mainly by Tyr86beta and N4' of ThDP. The hydrophobic moiety of MPA is recognized by Phe66alpha, Tyr95alpha, Met128alpha and His131alpha. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form. These results suggest that E1 undergoes an open-closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA. ES complex models of E1(holo).MOPA and of E1(ceim).lipoamide built from the above structures suggest that His273alpha and His129beta' are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively.
 ==About this Structure==
-UMD is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Thermus_thermophilus Thermus thermophilus] with MG, TDP and COI as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/3-methyl-2-oxobutanoate_dehydrogenase_(2-methylpropanoyl-transferring) 3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.2.4.4 1.2.4.4] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1UMD OCA].
+UMD is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Thermus_thermophilus Thermus thermophilus] with <scene name='pdbligand=MG:'>MG</scene>, <scene name='pdbligand=TDP:'>TDP</scene> and <scene name='pdbligand=COI:'>COI</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/3-methyl-2-oxobutanoate_dehydrogenase_(2-methylpropanoyl-transferring) 3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.2.4.4 1.2.4.4] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1UMD OCA].
 ==Reference==
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 [[Category: Nakagawa, N.]]
 [[Category: Nakai, T.]]
-[[Category: RSGI, RIKEN.Structural.Genomics/Proteomics.Initiative.]]
+[[Category: RSGI, RIKEN Structural Genomics/Proteomics Initiative.]]
 [[Category: COI]]
 [[Category: MG]]
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 [[Category: structural genomics]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 04:14:36 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:26:04 2008''