title: Direct demonstration of lipid phosphorylation in the lipid bilayer of the biomimetic bicontinuous cubic phase using the confined enzyme lipid A phosphoethanolamine transferase.
abstract: Retention of amphiphilic protein activity within the lipid bilayer membrane of the nanostructured biomimetic bicontinuous cubic phase is crucial for applications utilizing these hybrid protein-lipid self-assembly materials, such as in meso membrane protein crystallization and drug delivery. Previous work, mainly on soluble and membrane-associated enzymes, has shown that enzyme activity may be modified when immobilized, including membrane bound enzymes. The effect on activity may be even greater for amphiphilic enzymes with a large hydrophilic domain, such as the Neisserial enzyme lipid A phosphoethanolamine transferase (EptA). Encapsulation within the biomimetic but non-endogenous lipid bilayer membrane environment may modify the enzyme conformation, while confinement of the large hydrophilic domain with the nanoscale water channels of a continuous lipid bilayer structure may prevent full function of this enzyme. Herein we show that NmEptA remains active despite encapsulation within a nanostructured bicontinuous cubic phase. Full transfer of the phosphoethanolamine (PEA) group from a 1,2-dioleoyl-glycero-phosphoethanolamine (DOPE) doped lipid to monoolein (MO), which makes up the bicontinuous cubic phase, is shown. The reaction was found to be non-specific to the alkyl chain identity. The observed rate of enzyme activity is similar to other membrane bound enzymes, with complete transfer of the PEA group occurring in vitro, under the conditions studied, over a 24 hour timescale.
authors.affiliation: Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria 3010, Australia and CSIRO Manufacturing, Clayton, Victoria 3168, Australia.
authors.forename: Leonie
authors.identifier: http://orcid.org/0000-0003-1115-7524
authors.initials: L
authors.lastname: van 't Hag
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia 6009, Australia. alice.vrielink@uwa.edu.au.
authors.forename: Anandhi
authors.initials: A
authors.lastname: Anandan
authors.affiliation: CSIRO Manufacturing, Clayton, Victoria 3168, Australia.
authors.forename: Shane A
authors.initials: SA
authors.lastname: Seabrook
authors.affiliation: Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria 3010, Australia and The ARC Dairy Innovation Hub, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Sally L
authors.initials: SL
authors.lastname: Gras
authors.affiliation: CSIRO Manufacturing, Clayton, Victoria 3168, Australia and School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, Victoria 3001, Australia. charlotte.conn@rmit.edu.au.
authors.forename: Calum J
authors.identifier: http://orcid.org/0000-0001-7340-8611
authors.initials: CJ
authors.lastname: Drummond
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia 6009, Australia. alice.vrielink@uwa.edu.au.
authors.forename: Alice
authors.identifier: http://orcid.org/0000-0001-8197-3725
authors.initials: A
authors.lastname: Vrielink
authors.affiliation: School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, Victoria 3001, Australia. charlotte.conn@rmit.edu.au.
authors.forename: Charlotte E
authors.initials: CE
authors.lastname: Conn
mesh_terms: D004982:Ethanolaminephosphotransferase / Q000737:chemistry / Q000378:metabolism*; D008050:Lipid A / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009343:Neisseria / Q000201:enzymology; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation
chemical_list: D008050:Lipid A; D008051:Lipid Bilayers; D010714:Phosphatidylethanolamines; D004982:Ethanolaminephosphotransferase; C018525:1,2-dimyristoylphosphatidylethanolamine