Extended Data Fig. 6: Characterization of E. coli FabD transacylation activity with the DEBS PKS.
From: Engineering site-selective incorporation of fluorine into polyketides
(A) Steady-state kinetic characterization of transacylation reaction of malonyl-CoA (left), methylmalonyl-CoA (middle) and fluoromalonyl-CoA (right) catalyzed by FabD with 75 μM of ACPDEBSMod6 as acyl accepter. Activity was measured by α-ketoglutarate dehydrogenase coupled assay, monitoring coenzyme A release by NADH fluorescence. Curves were obtained from non-linear curve fitting of data to Hill equation modified for substrate inhibition model. Table contains kcat, K0.5, kcat/K0.5, Ki and n values. Data shown are mean ± s.d of three technical replicates. Error in kcat/KM is obtained by propagation from individual kinetic terms. (B) In vitro triketide lactone assay for FabD complementation of Mod3DEBS + TE(AT0). Normalized representation of the amount of F-TKL (left), H-TKL (middle) and TKL (right) products are shown for reactions containing 10 μM Mod3DEBS + TE(AT0)), 30 μM FabD or WT DszAT, 1 mM NDK-SNAC, 1 mM fluoromalonate (left), malonate (middle) or methylmalonate (right), and 1 mM coenzyme A under regenerative condition. Reactions were quenched and analyzed by LC-QQQ after 24 h. The amounts of products were determined by integrating extracted ion counts for the relevant species (transition): F-TKL (175 → 157), H-TKL (157 → 139) and TKL (171 → 153). Data are mean of two technical replicates.
