The 1.8 A crystal structure and active-site architecture of beta-ketoacyl-acyl carrier protein synthase III (FabH) from escherichia coli.
BACKGROUND
beta-Ketoacyl-acyl carrier protein synthase III (FabH) initiates elongation in type II fatty acid synthase systems found in bacteria and plants. FabH is a ubiquitous component of the type II system and is positioned ideally in the pathway to control the production of fatty acids. The elucidation of the structure of FabH is important for the understanding of its regulation by feedback inhibition and its interaction with drugs. Although the structures of two related condensing enzymes are known, the roles of the active-site residues have not been experimentally tested.


RESULTS
The 1.8 A crystal structure of FabH was determined using a 12-site selenium multiwavelength anomalous dispersion experiment. The active site (Cys112, His244 and Asn274) is formed by the convergence of two alpha helices and is accessed via a narrow hydrophobic tunnel. Hydrogen-bonding networks that include two tightly bound water molecules fix the positions of His244 and Asn274, which are critical for the decarboxylation and condensation reactions. Surprisingly, the His244-->Ala mutation does not affect the transacylation reaction suggesting that His244 has only a minor influence on the nucleophilicity of Cys112.


CONCLUSIONS
The histidine and asparagine active-site residues are both required for the decarboxylation step in the condensation reaction. The nucleophilicity of the active-site cysteine is enhanced by the alpha-helix dipole effect, and an oxyanion hole promotes the formation of the tetrahedral transition state.
Davies
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C
Department of Structural Biology, University of Tennessee, School of Biological Sciences, University of Sussex, Memphis, Falmer, 38163, BN1 9QG, USA, UK.
Heath
R J
RJ
White
S W
SW
Rock
C O
CO
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D017931:DNA Primers; C077402:3-ketoacyl-acyl carrier protein synthase III; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase