Abstract
Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the β-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N-acetyl transferase (GNAT) domain (MadAN) and a carboxy-terminal hot dog dimerization domain (MadAC) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis, we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadAC, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadAN did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.
Highlights
Bacterial type II fatty acid synthesis (FASII) is catalyzed by a collection of conserved enzymes that supply fatty acids for membrane phospholipid synthesis and has been the subject of intense study for decades [1]
MadB is a standalone hot dog malonyl-acyl carrier protein (ACP) decarboxylase from Shewanella oneidensis, and structure-guided mutagenesis identifies Asn45 as a key catalytic residue
We verify that glucosamine-1-phosphate N-acetyl transferase/N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU) is an ACP binding protein and show that acetyl-ACP is the preferred GlmU substrate for the synthesis of UDP-GlcNAc
Summary
Bacterial type II fatty acid synthesis (FASII) is catalyzed by a collection of conserved enzymes that supply fatty acids for membrane phospholipid synthesis and has been the subject of intense study for decades [1]. In 1987, an enzyme was discovered that catalyzes the condensation of acetyl-CoA with malonyl-ACP in Escherichia coli to initiate FASII [5], and the fabH gene encoding this activity was identified in 1992 [6]. Site-directed mutagenesis of the MadB catalytic loop residues produced properly folded proteins based on their thermal stabilities and dimeric structures (Table S3), and each was analyzed for Mad activity using the malonyl-CoA
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