Acetoacetyl-acyl carrier protein synthase. A target for the antibiotic thiolactomycin.
The biochemical basis for the inhibition of fatty acid biosynthesis in Escherichia coli by the antibiotic thiolactomycin was investigated. A biochemical assay was developed to measure acetoacetyl-acyl carrier protein (ACP) synthase activity, a recently discovered third condensing enzyme from E. coli (Jackowski, S., and Rock, C.O. (1987) J. Biol. Chem. 262, 7927-7931). In contrast to the other two condensing enzymes in E. coli, acetoacetyl-ACP synthase (synthase III) condensed malonyl-ACP with acetyl-CoA, rather than with acetyl-ACP. The concentration dependence of thiolactomycin inhibition of fatty acid biosynthesis in vivo was the same as the inhibition of acetoacetyl-ACP synthase activity in vitro indicating that the two phenomena were related. A thiolactomycin-resistant mutant (strain CDM5) was isolated. The specific activity of acetoacetyl-ACP synthase in extracts from this mutant was 10-fold lower than in extracts from its thiolactomycin-sensitive parent resulting in a marked defect in the ability of strain CDM5 to incorporate acetyl-CoA into fatty acids in vitro. The residual acetoacetyl-ACP synthase activity in the resistant strain was refractory to thiolactomycin inhibition. In addition, acetyl-CoA:ACP transacylase activity in strain CDM5 was resistant to inactivation by thiolactomycin suggesting that the acetoacetyl-ACP synthase also catalyzes this transacylation reaction. These data point to acetoacetyl-ACP synthase as a target for thiolactomycin inhibition of bacterial fatty acid biosynthesis.
Jackowski
S
S
Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
Murphy
C M
CM
Cronan
J E
JE
Rock
C O
CO
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000037:antagonists & inhibitors*; D000105:Acetyl Coenzyme A / Q000378:metabolism; D000213:Acyl Carrier Protein / Q000378:metabolism; D000217:Acyltransferases / Q000037:antagonists & inhibitors*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D005227:Fatty Acids / Q000096:biosynthesis*; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology; D008316:Malonyl Coenzyme A / Q000378:metabolism; D013876:Thiophenes
D000213:Acyl Carrier Protein; D005227:Fatty Acids; D007783:Lactones; D013876:Thiophenes; D008316:Malonyl Coenzyme A; D000105:Acetyl Coenzyme A; C035068:thiolactomycin; D000217:Acyltransferases; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase