Structure and mechanism of Staphylococcus aureus oleate hydratase (OhyA).
Bacterial Proteins
/ chemistry
Catalysis
Catalytic Domain
/ genetics
Crystallography, X-Ray
Fatty Acids, Unsaturated
/ chemistry
Humans
Hydro-Lyases
/ chemistry
Oleic Acid
/ chemistry
Protein Conformation
Staphylococcal Infections
/ enzymology
Staphylococcus aureus
/ chemistry
Substrate Specificity
/ genetics
Staphylococcus aureus (S. aureus)
X-ray crystallography
bacteria
enzyme catalysis
fatty acid metabolism
flavin adenine dinucleotide (FAD)
oleate hydratase (OhyA)
Journal
The Journal of biological chemistry
ISSN: 1083-351X
Titre abrégé: J Biol Chem
Pays: United States
ID NLM: 2985121R
Informations de publication
Date de publication:
Historique:
received:
17
11
2020
revised:
15
12
2020
accepted:
29
12
2020
pubmed:
31
12
2020
medline:
25
8
2021
entrez:
30
12
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 33376139
pii: S0021-9258(21)00019-3
doi: 10.1074/jbc.RA120.016818
pmc: PMC7948970
pii:
doi:
Substances chimiques
Bacterial Proteins
0
Fatty Acids, Unsaturated
0
Oleic Acid
2UMI9U37CP
Hydro-Lyases
EC 4.2.1.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
100252Subventions
Organisme : NCI NIH HHS
ID : P30 CA021765
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM034496
Pays : United States
Organisme : NIGMS NIH HHS
ID : R37 GM034496
Pays : United States
Organisme : NIAID NIH HHS
ID : T32 AI106700
Pays : United States
Informations de copyright
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
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