Flavin adenine dinucleotide (FAD)-dependent bacterial oleate hydratases (OhyAs) catalyze the addition of water to isolated fatty acid carbon–carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regiospecific and stereospecific hydration is required to understand their biological functions and the potential for engineering new products. In this study, we deduced the catalytic mechanism of OhyA from multiple structures of S. aureus OhyA in binary and ternary complexes with combinations of ligands along with biochemical analyses of relevant mutants. The substrate-free state shows Arg81 is the gatekeeper that controls fatty acid entrance to the active site. FAD binding engages the catalytic loop to simultaneously rotate Glu82 into its active conformation and Arg81 out of the hydrophobic substrate tunnel, allowing the fatty acid to rotate into the active site. FAD binding also dehydrates the active site, leaving a single water molecule connected to Glu82. This active site water is a hydronium ion based on the analysis of its hydrogen bond network in the OhyA•PEG400•FAD complex. We conclude that OhyA accelerates acid-catalyzed alkene hydration by positioning the fatty acid double bond to attack the active site hydronium ion, followed by the addition of water to the transient carbocation intermediate. Structural transitions within S. aureus OhyA channel oleate to the active site, curl oleate around the substrate water, and stabilize the hydroxylated product to inactivate antimicrobial fatty acids.

YfeA, a substrate-binding protein that is important for the virulence of Yersinia pestis, has two polyspecific metal-binding sites that may play different roles during infection. A flexible lobe at the carboxy-terminus suggests that structural rearrangement is required for metal transfer to binding partners.

Members of the Bacteroidetes phylum, represented by Alistipes finegoldii, are prominent anerobic, Gram‐negative inhabitants of the gut microbiome. The lipid biosynthetic pathways were analyzed using bioinformatic analyses, lipidomics, metabolic labeling and biochemistry to characterize exogenous fatty acid metabolism. A. finegoldii only produced the saturated fatty acids. The most abundant lipids were phosphatidylethanolamine (PE) and sulfonolipid (SL). Neither phosphatidylglycerol nor cardiolipin are present. PE synthesis is initiated by the PlsX/PlsY/PlsC pathway, whereas the SL pathway is related to sphingolipid biosynthesis. A. finegoldii incorporated medium‐chain fatty acids (≤14 carbons) into PE and SL after their elongation, whereas long‐chain fatty acids (≥16 carbons) were not elongated. Fatty acids >16 carbons were primarily incorporated into the 2‐position of phosphatidylethanolamine at the PlsC step, the only biosynthetic enzyme that utilizes long‐chain acyl‐ACP. The ability to assimilate a broad‐spectrum of fatty acid chain lengths present in the gut environment is due to the expression of two acyl‐acyl carrier protein (ACP) synthetases. Acyl‐ACP synthetase 1 had a substrate preference for medium‐chain fatty acids and synthetase 2 had a substrate preference for long‐chain fatty acids. This unique combination of synthetases allows A. finegoldii to utilize both the medium‐ and long‐chain fatty acid nutrients available in the gut environment to assemble its membrane lipids.

The oleate hydratase protein family was discovered in commensal bacteria that utilize host unsaturated fatty acids as the substrates to produce a spectrum of hydroxylated products. These hydroxy fatty acids are thought to act as signaling molecules that suppress the inflammatory response to create a more tolerant environment for the microbiome. S. aureus is a significant human pathogen, and defining the mechanisms used to evade the immune response is critical to understanding pathogenesis. S. aureus expresses an OhyA that produces at least three 10-hydroxy fatty acids from host unsaturated fatty acids at the infection site, and an S. aureus strain lacking the ohyA gene has compromised virulence in an immunocompetent infection model.

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 Δ fabH 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 m alonyl- A CP d ecarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N -acetyl transferase (GNAT) domain (MadA N ) and a carboxy-terminal hot dog dimerization domain (MadA C ) 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, MadA C , or MadB expression all restored normal cell size and growth rates to an E. coli Δ fabH strain, whereas the expression of MadA N 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.