Structure and mechanism of Staphylococcus aureus oleate hydratase (OhyA)

Abstract

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.

Enterococcus faecalis Readily Adapts Membrane Phospholipid Composition to Environmental and Genetic Perturbation

“…Even if oleic acid is not used well by the Fak system in a mprF2 deletion background, we would expect to see it accumulate free in the membrane, and that would be reflected in our GC-FAME analysis. Another possibility is that there is increased oleate hydratase activity in the gene deletion background, which would result in accumulation of C 18:0 , which could then enter fatty acid elongation cycle, generating C 20:0 (Schroepfer, 1966;Niehaus et al, 1970;Kishino et al, 2013;Radka et al, 2021). We do note an accumulation of C 20:0 in this strain, but also in other strains, upon supplementation with oleic acid (Supplementary Table 4).…”

Towards an understanding of oleate hydratases and their application in industrial processes

“…In Ohys, the FAD is non-covalently bound to the protein. Therefore, binding of FAD induces a conformational change in domain I, which leads to closure of the FAD-binding site and enfolding of the FAD [ 46 , 47 ].…”

Oleate Hydratase (OhyA) Is a Virulence Determinant in Staphylococcus aureus

“… Staphylococcus aureus is an important pathogen that deploys an array of virulence factors that engage host immune defenses to promote pathogenesis ( 6 – 8 ). S. aureus expresses an OhyA that catalyzes water addition to cis -9 double bonds ( 9 ) and protects against palmitoleic acid (16:1) ( 10 ), an antimicrobial fatty acid produced by the innate immune system ( 11 – 13 ). The goal of this study was to determine if OhyA has a role in S. aureus pathogenesis and supports the production of h FA at the infection site.…”