A Covalent Linker Allows for Membrane Targeting of an Oxylipin Biosynthetic Complex

Abstract

A naturally occurring bi-functional 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 8R-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 bi-functional 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 non-covalent 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 non-covalent interactions between the AOS and LOX domains and suggests that the C2-like domain of LOX mediates both protein-protein and protein-membrane interactions. KeywordsEicosanoids; lipoxygenase; allene oxide synthase; bi-functional enzymes; C2-domains; Calciumdependent membrane binding; arachidonic acid; protein-protein interactions; Small angle X-ray scattering (SAXS); X-ray crystallographyThe ability of the cell to respond to its environment is dependent upon effective coordination of metabolic pathways. For those pathways that involve the biosynthesis of the arachidonic acid (AA) -derived lipid mediators such as the leukotrienes, prostaglandins, and thromboxanes, their coordination may be exceptionally challenging in terms of substrate acquisition and specificity. The hydrophobic substrates partition into the membrane phase, and active sites that recognize bulky hydrophobic compounds might be inherently promiscuous. Furthermore, the highly reactive fatty acid hydroperoxide intermediates promote cellular oxidative damage if their metabolism is not stringently regulated. Both compartmentalization of enzymes and organization of multi-enzyme complexes are thought to provide cellular mechanisms for "traffic control" in pathways for the synthesis of these potent signaling molecules (1-3). In order to understand how coordination of biosynthetic pathways is achieved in the context of *Author to whom correspondence should be addressed: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, 70803, Tel : (225) Fax: (225) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript cell trafficking, and specifically how facilitated transfer of intermediates between active sites might be a means to regulate p...

Structural and functional insights into the reaction specificity of catalase-related hydroperoxide lyase: A shift from lyase activity to allene oxide synthase by site-directed mutagenesis

“…In parallel, the cHPL R56G and I357V mutants eluted mostly as monomers ( Fig 5C and 5D ). Both substitutions, R56G and I357V, are located in the α-helical part of cAOS which interacts with the LOX domain of the fusion protein [ 32 ]. Presumably, G56 and V357 influenced the properties of the corresponding α-helices and therefore, the oligomerization of the cHPL domain was reduced.…”

A Catalase-related Hemoprotein in Coral Is Specialized for Synthesis of Short-chain Aldehydes

“…Illustrating the independence of both domains, the separately expressed P. homomalla AOS-LOX domains each retained activity (11), and a similar functional independence of the two domains of the cHPL-LOX was shown here. The covalent linkage of cHPL with 8R-LOX probably helps to target the fusion protein into membranes, assuring the close proximity of the component domains to the newly released arachidonic acid substrate, as demonstrated with cAOS-LOX (26).…”

ALLENE OXIDE SYNTHASE and HYDROPEROXIDE LYASE, Two Non-Canonical Cytochrome P450s in Arabidopsis thaliana and Their Different Roles in Plant Defense

“…An interesting precedent for the interaction of enzymes involved in oxylipin biosynthesis was provided by the soft coral Plexaura homomalla [57,58,59]. In this organism, a bifunctional LOX-AOS enzyme was identified in which AOS forms the NH 2 -terminal portion and 8 R -LOX the COOH-terminal portion of the polypeptide chain [59]. The two domains cooperate in oxylipin biosynthesis and catalyze consecutive steps in an eicosanoid-like biosynthetic pathway.…”