Beta-ketoacyl-acyl-carrier-protein synthase II
In the biosynthesis of fatty acids, the beta-ketoacyl-acyl carrier protein (ACP) synthases catalyse chain elongation by the addition of two-carbon units derived from malonyl-ACP to an acyl group bound to ACP.
In Escherichia coli , the chain elongation step of fatty acid biosynthesis is carried out by at least three different enzymes, KAS I, II and III. Both KAS I and KAS II catalyse the condensation of a wide range of saturated acyl-ACPs. KAS I, however, may be responsible for a condensation reaction that cannot be catalysed by KAS II, namely elongation of C10:1, whereas KAS II is responsible for the elongation of palmoleitic acid C16:1 to cis -vaccenic acid C18:1. In E.coli, cis vaccenic acid synthesis increases upon a temperature downshift, and KAS II is believed to play a key role in this thermal regulation of fatty acid biosynthesis. KAS II is a homodimer and also known as FabF, name of gene coding for it.
.Reference Protein and Structure
- Sequence
-
P0AAI5
(2.3.1.179)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Escherichia coli K-12 (Bacteria)
- PDB
-
1kas
- BETA-KETOACYL-ACP SYNTHASE II FROM ESCHERICHIA COLI
(2.4 Å)
- Catalytic CATH Domains
-
3.40.47.10
(see all for 1kas)
Enzyme Reaction (EC:2.3.1.179)
Enzyme Mechanism
Introduction
The reaction can be described by three distinct steps: (i) the acyl group of acyl-ACP is transferred to a cysteine residue at the active site of the enzyme resulting in a thioester, developing negative charges on carbonyl in acyl-ACP stabilised by amides on Cys163 and Phe392 backbone ; (ii) generation of a carbanion by the elimination of bicarbonate from malonyl-ACP which is facilitated by His303 and stabilised by His340; and (iii) carbon-carbon bond formation by nucleophilic attack of the carbanion onto the carbonyl carbon atom of the thioester, stabilised by the backbone amides of Cys163 and Phe400. In chemical terms, the overall reaction can be classified as a Claisen condensation.
Catalytic Residues Roles
| UniProt | PDB* (1kas) | ||
| Cys164 | Cys163A | Acts as nucleophile to attack the carbonyl group in acyl-ACP, subsequent collapse of tetrahedral intermediate yields a thioester. Further reaction steps take place on the thioester intermediate. In the final step attack of a carbanion on the carbonyl group, the collapse of this tetrahedral intermediate cleaves the substrate enzyme bond releasing the cysteine residue. Note the thiolate form of Cys163 is not generated by His residues, instead it is stabilised by the helix dipole that is generate at the N terminus of the alpha-helix in the nucleophilic elbow. Also the backbone amide of Cys163 then forms an oxyanion hole to stabilise the build up of negative charge following attack, and again during the attack of the carbanion on the acyl-enzyme intermediate. | covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor, electrostatic stabiliser |
| Phe401 (main-N) | Phe400A (main-N) | Acts to stabilise build up of negative charge during initial nucleophilic attack via the formation of an oxyanion hole, and again in the attack of the carbanion on the acyl enzyme intermediate. | electrostatic stabiliser |
| His304 | His303A | Abstracts a proton from a water molecule to facilitate attack on the malonyl carboxylate, aided in this function by Lys325 and Glu314. | activator, proton acceptor, proton donor |
| Glu315 | Glu314A | Forms part of hydrogen bonding network to maintain the lone pair on the N-eta atom of His303. | electrostatic stabiliser |
| Lys336 | Lys335A | Forms a hydrogen bond to His303 to keep the lone pair of electrons on the N-eta atom on the imidazole ring. | electrostatic stabiliser |
| His341 | His340A | Lone pair on N-delta and protonates N-eta nitrogen serves to stabilise the negative charge developing on the malonyl thioester carbonyl. | hydrogen bond donor, electrostatic stabiliser |
Chemical Components
bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex formation, intermediate formation, overall reactant used, inferred reaction step, keto-enol tautomerisation, overall product formed, electron relay, intermediate collapse, enzyme-substrate complex cleavage, intermediate terminated, claisen condensation, native state of enzyme regeneratedReferences
- White SW et al. (2005), Annu Rev Biochem, 74, 791-831. THE STRUCTURAL BIOLOGY OF TYPE II FATTY ACID BIOSYNTHESIS. DOI:10.1146/annurev.biochem.74.082803.133524. PMID:15952903.
Step 1. Cys163 activated by the helix dipole, attacks the carbonyl group on the acyl-ACP. Inferred proton transferred steps.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Cys163A | covalently attached, electrostatic stabiliser |
| Phe400A (main-N) | electrostatic stabiliser |
| Lys335A | electrostatic stabiliser |
| Glu314A | electrostatic stabiliser |
| His340A | electrostatic stabiliser |
| Cys163A | proton donor, nucleophile |
Chemical Components
ingold: bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex formation, intermediate formation, overall reactant used, inferred reaction step
Step 2. Enol intermediate formation from His303 activated water attacking terminal carboxylate. His303 subsequently stabilises this intermediate, resulting in release of bicarbonate (in turn can become carbon dioxide).
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Cys163A | covalently attached |
| His303A | activator |
| His340A | hydrogen bond donor, electrostatic stabiliser |
| Lys335A | electrostatic stabiliser |
| Glu314A | electrostatic stabiliser |
| His303A | proton acceptor |
Chemical Components
ingold: bimolecular nucleophilic substitution, proton transfer, intermediate formation, keto-enol tautomerisation, overall product formed, overall reactant used
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| His340A | electrostatic stabiliser |
| Cys163A | covalently attached |
Chemical Components
electron relay, intermediate collapse
Step 4. Carboanion attack on the malonyl-ACP to form the product β-ketoacyl-ACP.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Cys163A | electrostatic stabiliser |
| Phe400A (main-N) | electrostatic stabiliser |
| Cys163A | nucleofuge |
Chemical Components
ingold: bimolecular nucleophilic substitution, enzyme-substrate complex cleavage, overall product formed, intermediate terminated, claisen condensation
Step 5. Proton transfer to regenerate active site for another round of catalysis.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Cys163A | proton acceptor |
| His303A | proton donor |
Download: