PDBsum entry: 2alm

Transferase

PDB id: 2alm

Contents

Protein chain

412 a.a. *

Metals

_MG ×2

Waters ×40

* Residue conservation analysis

PDB id:

2alm

Name:

Transferase

Title:

Crystal structure analysis of a mutant beta-ketoacyl-[acyl c protein] synthase ii from streptococcus pneumoniae

Structure:

3-oxoacyl-(acyl-carrier-protein) synthase ii. Chain: a. Engineered: yes. Mutation: yes

Source:

Streptococcus pneumoniae. Organism_taxid: 1313. Gene: fabf. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.60Å R-factor: 0.195 R-free: 0.257

Authors:

Y.M.Zhang,J.Hurlbert,S.W.White,C.O.Rock

Key ref:

Y.M.Zhang et al. (2006). Roles of the active site water, histidine 303, and phenylalanine 396 in the catalytic mechanism of the elongation condensing enzyme of Streptococcus pneumoniae. J Biol Chem, 281, 17390-17399. PubMed id: 16618705 DOI: 10.1074/jbc.M513199200

Date:

07-Aug-05 Release date: 30-Aug-05

Protein chain

Q9FBC2  (Q9FBC2_STRPN) -  3-oxoacyl-[acyl-carrier-protein] synthase 2

Seq: 411 a.a.

Struc: 412 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 

• Biological process: metabolic process (4 terms)
• Biochemical function: catalytic activity (6 terms)

DOI no: 10.1074/jbc.M513199200

J Biol Chem 281:17390-17399 (2006)

PubMed id: 16618705

Roles of the active site water, histidine 303, and phenylalanine 396 in the catalytic mechanism of the elongation condensing enzyme of Streptococcus pneumoniae.

Y.M.Zhang, J.Hurlbert, S.W.White, C.O.Rock.

ABSTRACT

beta-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (beta-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. The two active site histidines in the elongation condensing enzyme have different electronic states and functions. His337 is essential for condensation activity, and its protonated Nepsilon stabilizes the negative charge developed on the malonyl thioester carbonyl in the transition state. The Nepsilon of His303 accelerated catalysis by deprotonating a structured active site water for nucleophilic attack on the C3 of malonate, releasing bicarbonate. Lys332 controls the electronic state of His303 and also plays a critical role in the positioning of His337. Phe396 functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes.

Selected figure(s)

Figure 5.
Structures of the SpFabF[H303A] and the FabF-cerulenin binary complex. A, the structure of the SpFabF[H303A] mutant active site. The His^303 imidazole ring is missing, leading to the absence of structured waters within the active site and the free rotation of the Phe^396 side chain. The structure of the native protein is shown in translucent green. B, structure of the FabF-cerulenin binary complex (accession number 1B3N) (41) compared with the free enzyme. Cerulenin forms a covalent derivative with the active site cysteine, and its hydrophobic tail extends into the acyl chain binding pocket of the enzyme. Numbering of residues is according to the EcFabF sequence. The orientation of Phe^400 (Phe^396 in SpFabF) in the free enzyme is shown in translucent green to illustrate the movement of this side chain when the active site cysteine is acylated.

Figure 6.
Proposed mechanism for the elongation condensing enzymes. The first step involves the binding of acyl-ACP and the transfer of the acyl group to the active site cysteine. The nucleophilicity of Cys^164 is enhanced by the helix dipole effect, and the oxyanion hole formed by the backbone amides of Phe^396 and Cys^164 promotes the reaction by neutralizing the negative charge on the thioester carbonyl that develops in the transition state. ACP is released, and malonyl-ACP binds to the enzyme. His^303 activates a catalytic water molecule to attack the carboxylate of the malonyl-ACP and release bicarbonate. His^337 promotes the formation of the carbanion at C2 of the malonate by stabilizing the enol intermediate. The carbanion attacks the acyl-enzyme intermediate, and the tetrahedral transition state is stabilized by the Cys^164-Phe^396 oxyanion hole. The transition state resolves to form the β-ketoacyl-ACP product.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 17390-17399) copyright 2006.

Figures were selected by an automated process.