It is known that FabH transfers the acetyl group of acetyl-CoA onto its active site Cys112 followed by decarboxylation of malonyl-S-ACP forming an enolate intermediate that reacts with acetyl-S-FabH generating acetoacetyl-S-ACP, Figure . Mutation of the FabH active site cysteine to a glutamine is expected to generate a malonyl-S-ACP decarboxylase, similar to other β-ketoacyl-ACP synthases, Figure and can enable studies of the decarboxylation reaction. Structures of apo-FabH reveal many active site loops can be disordered and become ordered upon substrate presentation. Subsequent, studies probing FabH structure and catalysis with CoA mixed disulfides revealed negative cooperativity between the active sites of the dimer. We expect that substrate analogs combined with structural biology can reveal the missing aspects of negative cooperativity, conformational changes and decarboxylation mechanism, but a first step is synthesis and characterization of substrate analog:enzyme affinity and stability.