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 α-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.