title: A rapid fluorometric assay for the S-malonyltransacylase FabD and other sulfhydryl utilizing enzymes.
abstract: The development of biorenewable chemicals will support green chemistry initiatives and supplement the catalog of starting materials available to the chemical industry. Bacterial fatty acid biosynthesis is being pursued as a source of protein catalysts to synthesize novel reduced carbon molecules in fermentation systems. The availability of methods to measure enzyme catalysis for native and engineered enzymes from this pathway remains a bottleneck because a simple quantitative enzyme assay for numerous enzymes does not exist. Here we present two variations of a fluorescence assay that is readily extendable to high-throughput screening and is appropriate for thiol consuming and generating enzymes including theEscherichiacoli enzymes malonyl-coenzyme A transacylase (FabD) and keto-acylsynthase III (FabH). We measuredKMvalues of 60 ± 20 µM (acetyl-CoA) and 20 ± 10 µM (malonyl-ACP) and akcatof 7.4-9.0 s-1with FabH. Assays of FabD included a precipitation step to remove the thiol-containing substrate holo-ACP from the reaction product coenzyme-A to estimate reaction rates. Analysis of initial velocity measurements revealedKMvalues of 60 ± 20 µM (malonyl-CoA) and 40 ± 10 µM (holo-ACP) and akcatof 2100-2600 s-1for the FabD enzyme. Our data show similar results when compared to existing radioactive and continuous coupled assays in terms of sensitivity while providing the benefit of simplicity, scalability and repeatability. Fluorescence detection also eliminates the need for radioactive substrates traditionally used to assay these enzymes.
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Molecular Biology Building, Room 4210, 2437 Pammel Drive, Iowa State University, Ames, IA 50011, USA.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Marcella
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Molecular Biology Building, Room 4210, 2437 Pammel Drive, Iowa State University, Ames, IA 50011, USA.
authors.forename: Adam W
authors.initials: AW
authors.lastname: Barb
keywords: CPM; FabD; FabH; enzyme kinetics; high throughput