Dissecting the biophysical mechanisms of oleate hydratase association with membranes
This study investigates the dynamics of oleate hydratase (OhyA), a bacterial flavoenzyme from Staphylococcus aureus, and its interactions with lipid membranes, focusing on the factors influencing membrane binding and oligomerization. OhyA catalyzes the hydration of unsaturated fatty acids, playing a...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-01-01
|
| Series: | Frontiers in Molecular Biosciences |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2024.1504373/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1841555113020227584 |
|---|---|
| author | William A. Lathram Robert J. Neff Ashley N. Zalla James D. Brien Vivekanandan Subramanian Christopher D. Radka |
| author_facet | William A. Lathram Robert J. Neff Ashley N. Zalla James D. Brien Vivekanandan Subramanian Christopher D. Radka |
| author_sort | William A. Lathram |
| collection | DOAJ |
| description | This study investigates the dynamics of oleate hydratase (OhyA), a bacterial flavoenzyme from Staphylococcus aureus, and its interactions with lipid membranes, focusing on the factors influencing membrane binding and oligomerization. OhyA catalyzes the hydration of unsaturated fatty acids, playing a key role in bacterial pathogenesis by neutralizing host antimicrobial fatty acids. OhyA binds the membrane bilayer to access membrane-embedded substrates for catalysis, and structural studies have revealed that OhyA forms oligomers on membrane surfaces, stabilized by both protein-protein and protein-lipid interactions. Using fluorescence correlation spectroscopy (FCS), we examined the effects of membrane curvature and lipid availability on OhyA binding to phosphatidylglycerol unilamellar vesicles. Our results reveal that OhyA preferentially binds to vesicles with moderate curvature, while the presence of substrate fatty acids slightly enhanced the overall interaction despite reducing the binding affinity by 3- to 4-fold. Complementary phosphorus-31 (31P) NMR spectroscopy further demonstrated two distinct binding modes: a fast-exchange interaction at lower protein concentrations and a longer lasting interaction at higher protein concentrations, likely reflecting cooperative oligomerization. These findings highlight the reversible, non-stoichiometric nature of OhyA•membrane interactions, with dynamic binding behaviors influenced by protein concentration and lipid environment. This research provides new insights into the dynamic behavior of OhyA on bacterial membranes, highlighting that initial interactions are driven by lipid-mediated protein binding, while sustained interactions are primarily governed by the protein:lipid molar ratio rather than the formation of new, specific lipid-protein interactions. These findings advance our understanding of the biophysical principles underlying OhyA’s role in bacterial membrane function and virulence. |
| format | Article |
| id | doaj-art-b1073470a0cd4e13b9523e4c3d30a4f6 |
| institution | Kabale University |
| issn | 2296-889X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj-art-b1073470a0cd4e13b9523e4c3d30a4f62025-01-08T06:11:58ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2025-01-011110.3389/fmolb.2024.15043731504373Dissecting the biophysical mechanisms of oleate hydratase association with membranesWilliam A. Lathram0Robert J. Neff1Ashley N. Zalla2James D. Brien3Vivekanandan Subramanian4Christopher D. Radka5Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United StatesDepartment of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United StatesDepartment of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United StatesDepartment of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United StatesDepartment of Pharmaceutical Sciences, University of Kentucky, Lexington, KY, United StatesDepartment of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United StatesThis study investigates the dynamics of oleate hydratase (OhyA), a bacterial flavoenzyme from Staphylococcus aureus, and its interactions with lipid membranes, focusing on the factors influencing membrane binding and oligomerization. OhyA catalyzes the hydration of unsaturated fatty acids, playing a key role in bacterial pathogenesis by neutralizing host antimicrobial fatty acids. OhyA binds the membrane bilayer to access membrane-embedded substrates for catalysis, and structural studies have revealed that OhyA forms oligomers on membrane surfaces, stabilized by both protein-protein and protein-lipid interactions. Using fluorescence correlation spectroscopy (FCS), we examined the effects of membrane curvature and lipid availability on OhyA binding to phosphatidylglycerol unilamellar vesicles. Our results reveal that OhyA preferentially binds to vesicles with moderate curvature, while the presence of substrate fatty acids slightly enhanced the overall interaction despite reducing the binding affinity by 3- to 4-fold. Complementary phosphorus-31 (31P) NMR spectroscopy further demonstrated two distinct binding modes: a fast-exchange interaction at lower protein concentrations and a longer lasting interaction at higher protein concentrations, likely reflecting cooperative oligomerization. These findings highlight the reversible, non-stoichiometric nature of OhyA•membrane interactions, with dynamic binding behaviors influenced by protein concentration and lipid environment. This research provides new insights into the dynamic behavior of OhyA on bacterial membranes, highlighting that initial interactions are driven by lipid-mediated protein binding, while sustained interactions are primarily governed by the protein:lipid molar ratio rather than the formation of new, specific lipid-protein interactions. These findings advance our understanding of the biophysical principles underlying OhyA’s role in bacterial membrane function and virulence.https://www.frontiersin.org/articles/10.3389/fmolb.2024.1504373/fulloleate hydratase (OhyA)phospholipidmembrane bilayerfluorescence correlation spectroscopy (FCS)phosphorus nuclear magnetic resonance (31P NMR)membrane binding |
| spellingShingle | William A. Lathram Robert J. Neff Ashley N. Zalla James D. Brien Vivekanandan Subramanian Christopher D. Radka Dissecting the biophysical mechanisms of oleate hydratase association with membranes Frontiers in Molecular Biosciences oleate hydratase (OhyA) phospholipid membrane bilayer fluorescence correlation spectroscopy (FCS) phosphorus nuclear magnetic resonance (31P NMR) membrane binding |
| title | Dissecting the biophysical mechanisms of oleate hydratase association with membranes |
| title_full | Dissecting the biophysical mechanisms of oleate hydratase association with membranes |
| title_fullStr | Dissecting the biophysical mechanisms of oleate hydratase association with membranes |
| title_full_unstemmed | Dissecting the biophysical mechanisms of oleate hydratase association with membranes |
| title_short | Dissecting the biophysical mechanisms of oleate hydratase association with membranes |
| title_sort | dissecting the biophysical mechanisms of oleate hydratase association with membranes |
| topic | oleate hydratase (OhyA) phospholipid membrane bilayer fluorescence correlation spectroscopy (FCS) phosphorus nuclear magnetic resonance (31P NMR) membrane binding |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2024.1504373/full |
| work_keys_str_mv | AT williamalathram dissectingthebiophysicalmechanismsofoleatehydrataseassociationwithmembranes AT robertjneff dissectingthebiophysicalmechanismsofoleatehydrataseassociationwithmembranes AT ashleynzalla dissectingthebiophysicalmechanismsofoleatehydrataseassociationwithmembranes AT jamesdbrien dissectingthebiophysicalmechanismsofoleatehydrataseassociationwithmembranes AT vivekanandansubramanian dissectingthebiophysicalmechanismsofoleatehydrataseassociationwithmembranes AT christopherdradka dissectingthebiophysicalmechanismsofoleatehydrataseassociationwithmembranes |