Activity of fatty acid biosynthesis initiating ketosynthase FabH with acetyl/malonyl-oxa/aza(dethia)CoAs

Fatty acid biosynthetic enzymes exploit the reactivity of acyl- and malonyl-thioesters for catalysis. Here we synthesize acetyl/malonyl-CoA analogs with esters or amides in place of the thioester and characterize their behavior as substrates or inhibitors of the E. coli FabH ketosynthase. The acetyl- and malonyl-oxa(dethia)CoA analogs undergo extremely slow hydrolysis in the presence of FabH or C112Q mutant, which mimics the acyl-enzyme intermediate. Decarboxylation of malonyl-oxa(dethia)CoA by FabH or C112Q mutant was not detected. The amide analogs were completely stable to enzyme activity as expected. In enzyme assays, acetyl-oxa(dethia)CoA is surprisingly slightly activating, while acetyl-aza(dethia)CoA is a moderate inhibitor. The malonyl-oxa/aza(dethia)CoAs are inhibitors with Ki’s near the Km of malonyl-CoA. For comparison, we determine the FabH catalyzed decomposition rates for acetyl/malonyl-CoA, revealing some fundamental catalytic traits of FabH. The stable and inhibitory properties of the substrate analogs makes them promising for structure-function studies to undercover the basis of FabH cooperativity and enzyme:substrate interactions.