AOS

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Abstract

Allene oxide synthase (AOS) and 8R-lipoxygenase are parts of a naturally occurring fusion proteins in the coral Plexaura homomalla. AOS catalyzes the production of labile epoxide (an allene oxide) from fatty acid hydroperoxides. A study has reported the structure of the AOS domain and elucidated its significant structural homology to catalase. While the heme environment is largely conserved, AOS heme is planar with two hydrogen-bonded residues flanking the distal histidine. These key differences likely contributed to the switch from a catalatic activity to that of a fatty acid hydroperoxidase.

Introduction

Prostaglandins and other eicosanoids play an indispensable role in soft corals such as the Caribbean gorgonian Plexaura homomalla. Prostaglandins are formed from arachidonic acid via the conventional cyclooxygenase pathway, but the strong catalytic activity in coral extracts is lipoxygenase (LOX) pathway metabolism. Arachidonic acid is first converted to an 8R-hydroperoxide, which is then converted to allene oxide (epoxide). This pathway shares many similarities with the jasmonic acid biosynthesis route in plants (Fig. 1). In corals, this pathway may continue to produce cyclic products, but this connection remains to be proven. uring their study of the eicosanoid biosynthetic pathway in corals, Brash and coworkers identified a gene encoding a 122-kDa fusion protein that includes LOX and allene oxide synthase (AOS) domains. Its closest homologue in mammals is 5-LOX, an enzyme responsible for the synthesis of leukotrienes from arachidonic acid. This hemoglobin domain shares weak sequence identity to catalase, and all five homologous regions identified are involved in heme-binding or catalytic residues.

Spectroscopic data, including electron paramagnetic resonance, UV-vis, and magnetic CD, all indicate that AOS, like catalase, is a heme protein with a tyrosine axial ligand, but AOS does not have catalase active. Based on the sequence of plant AOS, the enzyme can be unambiguously identified as a member of the cytochrome P450 superfamily, that is, the member of a subfamily of the fatty acid hydroperoxide-metabolizing P450s designated as CYP74A. Other classes of P450 enzymes, such as mammalian prostacyclin synthase and thromboxane synthase, also catalyze similar chemical reactions in fatty acid peroxides. Therefore, coral AOS with sequence and spectral relationship to catalase is very specific.  

Figure 1. Comparison of allene oxide biosynthesis in coral and plants (Oldham, M.L.; et al. 2004)

The overall structure

The monomer of AOS is a wedge-shaped protein, approximately 45-50 Å in all three dimensions. The enzyme has an α+β fold consisting of an eight-stranded antiparallel β-barrel and 13 α-helices (Fig. 2). In the crystal, the monomers are associated through parallel interactions of the α3 helices (Fig. 3). This dimerization motif is observed in all four crystal forms (orthorhombic, triclinic, monoclinic, and C-centered monoclinic). Intermolecular contacts at the C-terminus involve the packing of threonines (T51, T54, and T58) in a fashion reminiscent of a leucine zipper. The oligomeric interface (1,070 Ų per monomer) is located at one edge of the wedge-shaped molecule, resulting in an overall bow tie-shaped dimer. Compared to typical heme proteins, the prosthetic group in AOS is very planar.

Figure 2. Stereo image of coral AOS backbone trace (Oldham, M.L.; et al. 2004)

Figure 3. The dimer interface formed by α3 of the two molecules in the asymmetric unit (Oldham, M.L.; et al. 2004)

AOS is present in a naturally occurring fusion protein

As mentioned earlier, coral AOS occurs as the N-terminal domain of the fusion protein, and the covalent fusion of AOS and LOX can enhance the mutual affinity of the two domains by increasing their effective concentration, thereby promoting the rapid transfer of the labile 8R-hydroperoxide from the LOX to the AOS active site. Marcotte et al. show that a fusion protein present in the genome of one species can be used to study protein-protein interactions in homologues in which the two proteins are expressed separately in other species.  Therefore, the fusion of AOS with LOX in corals may indicate an interaction between catalase-related AOS homologues and LOX in other organisms. Whether present in fusion proteins or as autonomous polypeptides, 8R-LOX and AOS activities are ubiquitous in corals, and their expression may be detected in other invertebrates.