Structural insights into the evolutionary paths of oxylipin biosynthetic enzymes

Abstract

The oxylipin pathway generates not only prostaglandin-like jasmonates but also green leaf volatiles (GLVs), which confer characteristic aromas to fruits and vegetables. Although allene oxide synthase (AOS) and hydroperoxide lyase are atypical cytochrome P450 family members involved in the synthesis of jasmonates and GLVs, respectively, it is unknown how these enzymes rearrange their hydroperoxide substrates into different products. Here we present the crystal structures of Arabidopsis thaliana AOS, free and in complex with substrate or intermediate analogues. The structures reveal an unusual active site poised to control the reactivity of an epoxyallylic radical and its cation by means of interactions with an aromatic pi-system. Replacing the amino acid involved in these steps by a non-polar residue markedly reduces AOS activity and, unexpectedly, is both necessary and sufficient for converting AOS into a GLV biosynthetic enzyme. Furthermore, by combining our structural data with bioinformatic and biochemical analyses, we have discovered previously unknown hydroperoxide lyase in plant growth-promoting rhizobacteria, AOS in coral, and epoxyalcohol synthase in amphioxus. These results indicate that oxylipin biosynthetic genes were present in the last common ancestor of plants and animals, but were subsequently lost in all metazoan lineages except Placozoa, Cnidaria and Cephalochordata.

Evidence for an Ionic Intermediate in the Transformation of Fatty Acid Hydroperoxide by a Catalase-related Allene Oxide Synthase from the Cyanobacterium Acaryochloris marina

“…Recently a P450 CYP74-catalyzed transformation of 13S-hydroperoxylinoleic acid to an 11-hydroxy-12,13-cis-epoxyalcohol was reported (13), which is more akin to our findings. In both cases the cis epoxide forms from the carbon bearing the hydroperoxide moiety.…”

“…The individual CYP74 enzymes include allene oxide synthase (AOS), one of which catalyzes a key step in cyclopentenone synthesis in the jasmonate pathway, hydroperoxide lyase, divinyl ether synthase, and epoxyalcohol synthase (12,13). The catalase-related enzymes are distinctive in that they have been found naturally as a fusion protein with the LOX enzyme that forms their hydroperoxide substrate (14).…”

Expression of Fusion Proteins of Aspergillus terreus Reveals a Novel Allene Oxide Synthase

“…The marked Asn residues of these class III P450 in Fig. 5 are important for this process (8,47). These two Asn residues align with Thr in P450cam and prostaglandin H 19-hydroxylase (CYP4F8) using ClustalW.…”

“…Allene oxides are precursors of JA, which is an important signal molecule in plant defense and development and is present throughout the plant kingdom (18). The P450 domain of LDS transforms 8R-HPODE to diols by intramolecular oxygen insertion following heterolytic cleavage of the hydroperoxide O-O bond with formation of compound I (Por ϩ ⅐ Fe(IV)ϭO) (8,16,17,19). The latter abstracts hydrogen at either C-7 or C-5 and catalyzes oxygen rebound with retention of the configuration (Fig.…”

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

“…The prototypical enzyme of this type is the cAOS-LOX fusion protein, which dioxygenates arachidonic acid to an 8R-hydroperoxyeicosatetraenoic acid (8R-HpETE) intermediate followed by its conversion to an allene oxide by the cAOS domain (11). Although the plant AOS and cAOS catalyze similar reactions, the enzymes are structurally unrelated (12)(13)(14). The highly labile allene oxide products of both enzyme types readily break down to stable end products, ␣-ketol and cyclopentenone (6).…”