A continuous coupled enzyme assay for bacterial malonyl-CoA:acyl carrier protein transacylase (FabD) |
| |
Authors: | Molnos Juliette Gardiner Rana Dale Glenn E Lange Roland |
| |
Institution: | Morphochem AG Basel, Schwarzwaldallee 215, Bldg. WRO-1055, CH-4058 Basel, Switzerland. |
| |
Abstract: | 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. |
| |
Keywords: | Fatty acid biosynthesis Malonyl-CoA:acyl carrier protein transacylase Coupled enzyme assay Acyl carrier protein Fluorescence |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|