2bui: Difference between revisions
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==Overview== | ==Overview== | ||
Beta-ketoacyl-acyl carrier protein (ACP) synthase enzymes join short | Beta-ketoacyl-acyl carrier protein (ACP) synthase enzymes join short carbon units to construct fatty acyl chains by a three-step Claisen condensation reaction. The reaction starts with a trans thioesterification of the acyl primer substrate from ACP to the enzyme. Subsequently, the donor substrate malonyl-ACP is decarboxylated to form a carbanion intermediate, which in the third step attacks C1 of the primer substrate giving rise to an elongated acyl chain. A subgroup of beta-ketoacyl-ACP synthases, including mitochondrial beta-ketoacyl-ACP synthase, bacterial plus plastid beta-ketoacyl-ACP synthases I and II, and a domain of human fatty acid synthase, have a Cys-His-His triad and also a completely conserved Lys in the active site. To examine the role of these residues in catalysis, H298Q, H298E and six K328 mutants of Escherichia colibeta-ketoacyl-ACP synthase I were constructed and their ability to carry out the trans thioesterification, decarboxylation and/or condensation steps of the reaction was ascertained. The crystal structures of wild-type and eight mutant enzymes with and/or without bound substrate were determined. The H298E enzyme shows residual decarboxylase activity in the pH range 6-8, whereas the H298Q enzyme appears to be completely decarboxylation deficient, showing that H298 serves as a catalytic base in the decarboxylation step. Lys328 has a dual role in catalysis: its charge influences acyl transfer to the active site Cys, and the steric restraint imposed on H333 is of critical importance for decarboxylation activity. This restraint makes H333 an obligate hydrogen bond donor at Nepsilon, directed only towards the active site and malonyl-ACP binding area in the fatty acid complex. | ||
==About this Structure== | ==About this Structure== | ||
2BUI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=NH4:'>NH4</scene> and <scene name='pdbligand=OCA:'>OCA</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. This structure | 2BUI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=NH4:'>NH4</scene> and <scene name='pdbligand=OCA:'>OCA</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. This structure supersedes the now removed PDB entry 1OG9. Active as [http://en.wikipedia.org/wiki/Beta-ketoacyl-acyl-carrier-protein_synthase_I Beta-ketoacyl-acyl-carrier-protein synthase I], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.1.41 2.3.1.41] Known structural/functional Site: <scene name='pdbsite=AC1:Oca+Binding+Site+For+Chain+D'>AC1</scene>. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2BUI OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Henriksen, A.]] | [[Category: Henriksen, A.]] | ||
[[Category: Olsen, J | [[Category: Olsen, J G.]] | ||
[[Category: Wettstein-Knowles, P | [[Category: Wettstein-Knowles, P V.]] | ||
[[Category: NH4]] | [[Category: NH4]] | ||
[[Category: OCA]] | [[Category: OCA]] | ||
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[[Category: transferase]] | [[Category: transferase]] | ||
''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 16:41:45 2008'' | ||
Revision as of 17:41, 21 February 2008
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E.COLI BETA-KETOACYL (ACYL CARRIER PROTEIN) SYNTHASE I IN COMPLEX WITH OCTANOIC ACID, 120K
OverviewOverview
Beta-ketoacyl-acyl carrier protein (ACP) synthase enzymes join short carbon units to construct fatty acyl chains by a three-step Claisen condensation reaction. The reaction starts with a trans thioesterification of the acyl primer substrate from ACP to the enzyme. Subsequently, the donor substrate malonyl-ACP is decarboxylated to form a carbanion intermediate, which in the third step attacks C1 of the primer substrate giving rise to an elongated acyl chain. A subgroup of beta-ketoacyl-ACP synthases, including mitochondrial beta-ketoacyl-ACP synthase, bacterial plus plastid beta-ketoacyl-ACP synthases I and II, and a domain of human fatty acid synthase, have a Cys-His-His triad and also a completely conserved Lys in the active site. To examine the role of these residues in catalysis, H298Q, H298E and six K328 mutants of Escherichia colibeta-ketoacyl-ACP synthase I were constructed and their ability to carry out the trans thioesterification, decarboxylation and/or condensation steps of the reaction was ascertained. The crystal structures of wild-type and eight mutant enzymes with and/or without bound substrate were determined. The H298E enzyme shows residual decarboxylase activity in the pH range 6-8, whereas the H298Q enzyme appears to be completely decarboxylation deficient, showing that H298 serves as a catalytic base in the decarboxylation step. Lys328 has a dual role in catalysis: its charge influences acyl transfer to the active site Cys, and the steric restraint imposed on H333 is of critical importance for decarboxylation activity. This restraint makes H333 an obligate hydrogen bond donor at Nepsilon, directed only towards the active site and malonyl-ACP binding area in the fatty acid complex.
About this StructureAbout this Structure
2BUI is a Single protein structure of sequence from Escherichia coli with and as ligands. This structure supersedes the now removed PDB entry 1OG9. Active as Beta-ketoacyl-acyl-carrier-protein synthase I, with EC number 2.3.1.41 Known structural/functional Site: . Full crystallographic information is available from OCA.
ReferenceReference
Fatty acid synthesis. Role of active site histidines and lysine in Cys-His-His-type beta-ketoacyl-acyl carrier protein synthases., von Wettstein-Knowles P, Olsen JG, McGuire KA, Henriksen A, FEBS J. 2006 Feb;273(4):695-710. PMID:16441657
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