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, 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.
Khandekar
S S
SS
Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA. Sanjay_Khandekar-1@sbphrd.com
Konstantinidis
A K
AK
Silverman
C
C
Janson
C A
CA
McNulty
D E
DE
Nwagwu
S
S
Van Aller
G S
GS
Doyle
M L
ML
Kane
J F
JF
Qiu
X
X
Lonsdale
J
J
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000105:Acetyl Coenzyme A / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012645:Selenomethionine / Q000737:chemistry* / Q000378:metabolism
D011994:Recombinant Proteins; D000105:Acetyl Coenzyme A; D012645:Selenomethionine; C077402:3-ketoacyl-acyl carrier protein synthase III; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase