Expression, purification, and crystallization of the Escherichia coli selenomethionyl beta-ketoacyl-acyl carrier protein synthase III

Bacterial beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, a lso called FabH) catalyzes the condensation and transacylation of acetyl-Co A with malonyl-ACP. In order to understand the mode of enzyme/substrate int eraction 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 HAS III was homog enous, stably folded, and enzymatically active. Dynamic light scattering, s ize exclusion chromatography, and mass spectrometry results indicated that selenomethionyl KAS III is a noncovalent homodimer. Diffraction quality cry stals of selenomethionyl HAS III/acetyl-CoA complex, which grew overnight t o a size of 0.2 mm(3), belonged to the tetragonal space group P4(1)2(1)2. ( C) 2000 Academic Press.