Initiation of Fatty Acid Synthesis by a Malonyl‐ACP Decarboxylase

Abstract

Fatty acid synthesis is important in both prokaryotes and eukaryotes for formation of functional membranes and dysregulation of the pathway has implications for cell growth and survival. In bacteria, fatty acid production is tightly regulated by the Type II fatty acid synthesis (FASII) pathway. In Escherichia coli FabH (3-ketoacyl acyl carrier protein (ACP) synthase III) initiates fatty acid synthesis by condensing acetyl coenzyme A (acetyl-CoA) with malonyl-ACP. This work describes a new pathway enzyme, malonyl-ACP decarboxylase (MadA), that permits ΔfabH knockout strains to survive by providing an alternate pathway for the initiation of fatty acid synthesis. MadA has an N-terminal GCN5-related N-acetyltransferase (GNAT) domain and a C-terminal hotdog domain. The full-length protein, the N-terminal (MadAN) and C-terminal (MadAC) domains were purified. MadA and MadAC are dimers and MadAN is a monomer. MadAC is a malonyl-ACP decarboxylase and MadAN is not. Mad proteins are widely distributed in Proteobacteria and most contain only the C-terminal hot dog dimerization and catalytic domain (MadB). The MadB homolog in Shewanella oneidensis was purified. Site directed mutagenesis guided by the MadB crystal structure was used to identify the active site loop. Asn45 has a critical role in the malonyl-ACP decarboxylase activity. MadA initiates FabH-independent fatty acid synthesis by decarboxylating malonyl-ACP to acetyl-ACP. FabB/F then condenses malonyl-ACP and acetyl-ACP to acetoacetyl-ACP, which enters the elongation cycle. MadAC and MadB both restore the cell size defect in the ΔfabH strain.