Beta-ketoacyl-[acyl carrier protein] synthase I
Beta-ketoacyl-acyl carrier protein synthase (KAS) I catalyses the Claisen condensation between acyl-ACP and malonyl-ACP to produce free acyl carrier protein (ACP), 3-oxoacyl ACP, and carbon dioxide. KAS I fuctions, together with KAS II and KAS III, to synthesise C16 and C18 fatty acids in plant plastids: KAS III is specific for the first step of the elongation and uses a CoA-activated primer substrate, while KAS I extends C4 to C16 in six rounds of elongation using an ACP substrate. KAS II then carries out an additional step to yield C18. In Escherichia coli, KAS I is essential for the construction of the unsaturated fatty acids characterising Escherichia coli membrane lipids.
Reference Protein and Structure
- Sequence
-
P0A953
(2.3.1.41)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Escherichia coli K-12 (Bacteria)
- PDB
-
1dd8
- CRYSTAL STRUCTURE OF BETA-KETOACYL-[ACYL CARRIER PROTEIN] SYNTHASE I FROM ESCHERICHIA COLI
(2.3 Å)
- Catalytic CATH Domains
-
3.40.47.10
(see all for 1dd8)
Enzyme Reaction (EC:2.3.1.41)
Enzyme Mechanism
Introduction
The catalysed reaction occurs in three steps. First, Cys 163 acts as a nucleophile to attack the carbonyl group of acyl ACP in a transesterification reaction that releases ACP and forms a Cys 163-linked thioester. The tetrahedral intermediate in this reaction is stabilised by an oxyanion hole composed of the backbone NH groups of Cys 163 and Phe 392. The nucleophilic nature of Cys 163 is enhanced by the positive end of an alpha-helix dipole. In the second step, His 298 deprotonates the carboxyl group of malonyl ACP to initiate the decarboxylation of this substrate with formation of an enolate intermediate. Accumulation of negative charge on the thioester carbonyl of malonyl CoA during formation of the enolate is stabilised by a hydrogen bond from His 333. The pKa of His 298 is modified by Lys 328 (acting indirectly via a hydroxide ion) and by a C-H...O hydrogen bond from the backbone oxygen of Phe 390. In the final step, the enolate produced from malonyl ACP attacks the Cys 163-linked thioester in a Claisen condensation reaction. This forms the product 3-oxoacyl-ACP and regenerates free Cys 163. The tetrahedral intermediate formed during the attack is stabilised by the oxyanion hole of Cys 163 and Phe 392.
Catalytic Residues Roles
| UniProt | PDB* (1dd8) | ||
| Lys328 | Lys328A | Indirectly modifies the pKa of His 298 by stabilising a hydroxide ion that interacts with the N-delta of His 298. | activator, hydrogen bond donor |
| His333 | His333A | Donates a hydrogen bond to the thioester carbonyl of maloyl-ACP, stabilising accumulation of negative charge on this atom during formation of the enolate intermediate. | increase basicity, hydrogen bond donor, electrostatic stabiliser |
| Phe390 (main-C) | Phe390A (main-C) | Modifies the pKa of His 298 by forming a C-H...O hydrogen bond from its backbone oxygen to C-epsilon of His 298. | activator, hydrogen bond acceptor |
| Phe392 (main-N) | Phe392A (main-N) | Backbone NH forms part of oxyanion hole that stabilises the tetrahedral intermediate that results from attack on the acyl-ACP carbonyl by the side chain of Cys 163 and later by the malonyl-ACP derived enolate. | hydrogen bond donor, electrostatic stabiliser |
| Cys163 | Cys163A | Attacks the thioester carbonyl of acyl ACP to form an enzyme-linked acyl thioester in a transesterification reaction. Backbone NH forms part of oxyanion hole that stabilises the tetrahedral intermediate that results from attack by the side chain of Cys 163 and later by the malonyl-ACP derived enolate. | covalently attached, hydrogen bond donor, nucleofuge, nucleophile, activator, electrostatic stabiliser |
| His298 | His298A | Deprotonates the carboxyl group of malonyl ACP to initiate the decarboxylation reaction. Backbone oxygen forms a C-H...O hydrogen bond to C-epsilon of His 298 which modifies the pKa of the imidazole ring. | hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser |
Chemical Components
bimolecular nucleophilic addition, overall reactant used, intermediate formation, enzyme-substrate complex formation, unimolecular elimination by the conjugate base, intermediate collapse, enzyme-substrate complex cleavage, proton transfer, overall product formed, decarboxylation, claisen condensation, native state of enzyme regenerated, inferred reaction stepReferences
- Olsen JG et al. (2001), Structure, 9, 233-243. Structures of β-Ketoacyl-Acyl Carrier Protein Synthase I Complexed with Fatty Acids Elucidate its Catalytic Machinery. DOI:10.1016/s0969-2126(01)00583-4. PMID:11286890.
- McGuire KA et al. (2001), Biochemistry, 40, 9836-9845. β-Ketoacyl-[Acyl Carrier Protein] Synthase I ofEscherichia coli: Aspects of the Condensation Mechanism Revealed by Analyses of Mutations in the Active Site Pocket†. DOI:10.1021/bi0105577. PMID:11502177.
- Clark JD et al. (1988), Biochemistry, 27, 5961-5971. Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test. DOI:10.1021/bi00416a020. PMID:2847778.
Step 1. The side chain thiolate of Cys163 acts as a nucleophile towards the carbonyl group of acyl ACP in a transesterification reaction that forms a Cys163-linked tetrahedral anion intermediate. The positive end of an alpha helix dipole enhances the nucleophilic character of Cys163 by stabilising the anionic thiolate form [PMID:11286890].
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Cys163A | hydrogen bond donor, electrostatic stabiliser |
| Phe392A (main-N) | hydrogen bond donor, electrostatic stabiliser |
| His298A | hydrogen bond donor, hydrogen bond acceptor |
| Lys328A | hydrogen bond donor |
| Phe390A (main-C) | hydrogen bond acceptor |
| Cys163A | nucleophile |
Chemical Components
ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation, enzyme-substrate complex formation
Step 2. The tetrahedral intermediate collapses, releasing a terminal thiol anion form of ACP.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Cys163A | activator, covalently attached, hydrogen bond donor, electrostatic stabiliser |
| Phe392A (main-N) | hydrogen bond donor, electrostatic stabiliser |
| His298A | hydrogen bond donor, hydrogen bond acceptor |
| Lys328A | hydrogen bond donor |
| Phe390A (main-C) | hydrogen bond acceptor |
Chemical Components
ingold: unimolecular elimination by the conjugate base, intermediate collapse, intermediate formation, enzyme-substrate complex cleavage
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Cys163A | covalently attached, hydrogen bond donor |
| Phe392A (main-N) | hydrogen bond donor |
| His298A | hydrogen bond donor, hydrogen bond acceptor |
| His333A | hydrogen bond donor, increase basicity |
| Lys328A | hydrogen bond donor, activator |
| Phe390A (main-C) | hydrogen bond acceptor, activator |
| His298A | proton acceptor |
Chemical Components
proton transfer, intermediate formation, overall reactant used
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Cys163A | covalently attached, hydrogen bond donor |
| Phe392A (main-N) | hydrogen bond donor |
| His298A | hydrogen bond donor, electrostatic stabiliser |
| His333A | hydrogen bond donor, electrostatic stabiliser |
| Lys328A | hydrogen bond donor, activator |
| Phe390A (main-C) | hydrogen bond acceptor, activator |
Chemical Components
ingold: unimolecular elimination by the conjugate base, overall product formed, intermediate formation, decarboxylation
Step 5. The ACP bound acetyl carbanion attacks the thioester carbon of the bound fatty acid, and a second tetrahedral intermediate is formed. Accumulation of negative charge on the thioester carbonyl of malonyl CoA during formation of the enolate is stabilised by a hydrogen bond from His 333. The pKa of His298 is modified by Lys328 (acting indirectly via a hydroxide ion) and by a C-H---O hydrogen bond from the backbone oxygen of Phe390 [PMID:11502177].
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Cys163A | activator, covalently attached, hydrogen bond donor, electrostatic stabiliser |
| Phe392A (main-N) | hydrogen bond donor, electrostatic stabiliser |
| His298A | hydrogen bond donor, electrostatic stabiliser |
| His333A | hydrogen bond donor, electrostatic stabiliser |
| Lys328A | hydrogen bond donor, activator |
| Phe390A (main-C) | hydrogen bond acceptor, activator |
Chemical Components
ingold: bimolecular nucleophilic addition, claisen condensation, intermediate formation, enzyme-substrate complex formation
Step 6. The tetrahedral intermediate collapses, eliminating the Cys163 thiolate and generating an extended carbon chain, with an acyl-carrier protein component still attached.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Cys163A | covalently attached, hydrogen bond donor, electrostatic stabiliser |
| Phe392A (main-N) | hydrogen bond donor, electrostatic stabiliser |
| His298A | hydrogen bond donor, electrostatic stabiliser |
| His333A | hydrogen bond donor, electrostatic stabiliser |
| Lys328A | hydrogen bond donor, activator |
| Phe390A (main-C) | hydrogen bond acceptor, activator |
| Cys163A | nucleofuge |
Chemical Components
ingold: unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, enzyme-substrate complex cleavage
Step 7. The anionic form of ACP removes a proton from His298A, regenerating the active site for further catalysis.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| His298A | hydrogen bond donor |
| His333A | hydrogen bond donor |
| Lys328A | hydrogen bond donor, activator |
| Phe390A (main-C) | hydrogen bond acceptor, activator |
| His298A | proton donor |
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