A covalent linker allows for membrane targeting of an oxylipin biosynthetic complex.
A naturally occurring bifunctional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C-terminal lipoxygenase (LOX) portion of the molecule catalyzes the transformation of arachidonic acid (AA) to the corresponding 8 R-hydroperoxide, and the N-terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). Small-angle X-ray scattering data indicate that in the absence of a covalent linkage the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bifunctional protein. The SAXS data also support a model for LOX and AOS domain orientation in the fusion protein inferred from a low-resolution crystal structure. However, results of membrane binding experiments indicate that covalent linkage of the domains is required for Ca (2+)-dependent membrane targeting of the sequential activities, despite the noncovalent domain association. Furthermore, membrane targeting is accompanied by a conformational change as monitored by specific proteolysis of the linker that joins the AOS and LOX domains. Our data are consistent with a model in which Ca (2+)-dependent membrane binding relieves the noncovalent interactions between the AOS and LOX domains and suggests that the C2-like domain of LOX mediates both protein-protein and protein-membrane interactions.
Gilbert
Nathaniel C
NC
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Niebuhr
Marc
M
Tsuruta
Hiro
H
Bordelon
Tee
T
Ridderbusch
Oswin
O
Dassey
Adam
A
Brash
Alan R
AR
Bartlett
Sue G
SG
Newcomer
Marcia E
ME
D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D031541:Fluorescence Resonance Energy Transfer; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D008081:Liposomes; D011993:Recombinant Fusion Proteins; D008084:Lipoxygenase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase
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