A continuous coupled enzyme assay for bacterial malonyl-CoA:acyl carrier protein transacylase (FabD).
Bacterial malonyl-CoA:acyl carrier protein transacylase catalyzes the transfer of a malonyl moiety from malonyl-CoA to the free thiol group of the phosphopantetheine arm of acyl carrier protein. Malonyl-ACP, the product of this enzymatic reaction, is the key building block for de novo fatty acid biosynthesis. Here, we describe a continuous enzyme assay based on the coupling of the malonyl-CoA:acyl carrier protein transacylase reaction to alpha-ketoglutarate dehydrogenase (KDH). KDH-dependent consumption of the coenzyme A generated by malonyl-CoA:acyl carrier protein transacylase is accompanied by a reduction of nicotinamide adenine dinucleotide, oxidized (NAD(+)) to nicotinamide adenine dinucleotide, reduced. The rate of NAD(+) reduction is continuously monitored as a change in fluorescence using a microtiter plate reader. We show that this coupled enzyme assay is amenable to routine chemical compound screening.
Molnos
Juliette
J
Morphochem AG Basel, Schwarzwaldallee 215, Bldg. WRO-1055, CH-4058 Basel, Switzerland.
Gardiner
Rana
R
Dale
Glenn E
GE
Lange
Roland
R
D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism
D029968:Escherichia coli Proteins; D008316:Malonyl Coenzyme A; D007655:Ketoglutarate Dehydrogenase Complex; D000217:Acyltransferases; D051080:Acyl-Carrier Protein S-Malonyltransferase; C494968:fabD protein, E coli; D054889:Fatty Acid Synthase, Type II