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1.
Biochem Biophys Res Commun ; 270(1): 100-7, 2000 Apr 02.
Article in English | MEDLINE | ID: mdl-10733911

ABSTRACT

Bacterial beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called FabH) catalyzes the condensation and transacylation of acetyl-CoA with malonyl-ACP. In order to understand the mode of enzyme/substrate interaction and design small molecule inhibitors, we have expressed, purified, and crystallized a selenomethionyl-derivative of E. coli KAS III. Several lines of evidence confirmed that purified selenomethionyl KAS III was homogenous, stably folded, and enzymatically active. Dynamic light scattering, size exclusion chromatography, and mass spectrometry results indicated that selenomethionyl KAS III is a noncovalent homodimer. Diffraction quality crystals of selenomethionyl KAS III/acetyl-CoA complex, which grew overnight to a size of 0.2 mm(3), belonged to the tetragonal space group P4(1)2(1)2.


Subject(s)
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/chemistry , Acetyl Coenzyme A/chemistry , Escherichia coli/enzymology , Selenomethionine/chemistry , 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/biosynthesis , 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics , Chromatography, Gel , Circular Dichroism , Crystallization , Escherichia coli/genetics , Mass Spectrometry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Selenomethionine/metabolism
2.
J Biol Chem ; 274(51): 36465-71, 1999 Dec 17.
Article in English | MEDLINE | ID: mdl-10593943

ABSTRACT

Beta-ketoacyl-acyl carrier protein synthase III (FabH), the most divergent member of the family of condensing enzymes, is a key catalyst in bacterial fatty acid biosynthesis and a promising target for novel antibiotics. We report here the crystal structures of FabH determined in the presence and absence of acetyl-CoA. These structures display a fold that is common for condensing enzymes. The observed acetylation of Cys(112) proves its catalytic role and clearly defines the primer binding pocket. Modeling based on a bound CoA molecule suggests catalytic roles for His(244) and Asn(274). The structures provide the molecular basis for FabH substrate specificity and reaction mechanism and are important for structure-based design of novel antibiotics.


Subject(s)
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/chemistry , Bacterial Proteins/chemistry , Isoenzymes/chemistry , 3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/metabolism , Bacterial Proteins/metabolism , Escherichia coli , Fatty Acids/metabolism , Isoenzymes/metabolism , Molecular Sequence Data , Protein Conformation , Substrate Specificity
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