Tuberculosis remains a global health problem, and programs dedicated to discovery of novel compounds against Mycobacterium tuberculosis require robust assays for high-throughput screening of chemical and
natural product libraries.
Enzymes involved in the biosynthesis of
mycolic acids, vital components of the mycobacterial cell wall, have received much attention as potential
drug targets. KasA and KasB, examples of the
beta-ketoacyl-acyl carrier protein synthase I/II (KASI/II) class of condensing
enzymes of the M.
tuberculosis fatty acid synthase II system have been the focus of several studies designed to biochemically characterize these
enzymes. Whilst robust methods have been developed for FabH-like
proteins, fast and sensitive assays for high-throughput screening of KASI/II
enzymes have not been available. Here we report the development of a direct scintillation proximity assay (SPA) for the KASI/II
enzymes, KasA and KasB. The SPA was more sensitive than existing assays, as shown by its ability to measure activity using less
enzyme than other assay formats, and the SPA was validated using the known KAS inhibitor
thiolactomycin. In addition, the KasA and KasB SPA was adapted for use with Staphylococcus aureus FabF to show the versatility of this assay format to KAS
enzymes from other pathogenic organisms.