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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 π-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.

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Figure 1: Reactions catalysed by the CYP74 enzyme family.
Figure 2: Tertiary topology and substrate binding site of AOS.
Figure 3: Structural basis for evolving HPL activity from the AOS scaffold.
Figure 4: Proposed reaction paths for AOS and HPL on the basis of the current structural and enzymological studies14,15,16,17,21.
Figure 5: Discovery of CYP74 in bacteria and animals.
Figure 6: Common ancestry of oxylipin biosynthetic enzymes.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the RCSB Protein Data Bank under the following accession codes: 3CLI (At-AOS), 3DSI (At-AOS in complex with 13-HOT), 2RCH (At-AOS in complex with 13-HOD), 2RCL (At-AOS in complex with 12R,13S-vernolic acid), 2RCM (At-AOS(F137L)), 3DSJ (At-AOS(F137L) in complex with 13-HOD), and 3DSK (At-AOS(F137L) in complex with 12R,13S-vernolic acid). Nucleotide sequences of Ap-AOS, Bf-EAS and Mn-HPL have been deposited in Genbank under accession numbers EU541487, EU555186 and EU887514, respectively.

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Acknowledgements

C.S.R. is grateful to the late N. Natarajaratnam and the late T. Traylor for encouragement; R. Cudney for nonanoyl-N-hydroxyethylglucamide (HEGA-9); K. Matsui for tomato HPL complementary DNA; K. Back and D. Park for Arabidopsis and rice cDNA libraries; L. Holland and J. Langeland for the amphioxus cDNA library; M. Medina for A. palmata expressed sequence tags; C. Marx and L. Moulin for M. nodulans cells; R. Müller for S. aurantiaca genomic DNA; L. Roman and B. Masters for the pCWori+ vector; J. Navarro for the single crystal microspectrophotometer; T. Doukov, S. Soltis, A. Cohen and J. Charles for help with acquiring electronic absorption spectra of single crystals; S. Veeraraghavan for generating Fig. 5; R. Bach, T. Bach, A. Beckwith, W. Bernhard, D. Curran, A. Davies, T. Dibble, J. Finnerty, D. Fleischman, J. Froehlich, J. Groves, L. Holland, P. Holland, J. Howieson, H. Kaplan, D. Nelson, M. Newcomb, P. Ortiz de Montellano, N. Porter, T. Poulos, M. Sibi, S. Veeraraghavan and D. Whalen for discussions; Joint Genome Institute for access to sequence data; Stanford Synchrotron Radiation Laboratories (beam lines 9–2 and 11–1, T. Doukov and L. Dunn) and the Advanced Light Source (beam line 8.3.1, J. Holton, G. Meigs and J. Tanamachi; beam lines 8.2.1 and 8.2.2, C. Ralston) for beam time and assistance. This work is supported by Pew Charitable Trusts through a Pew Scholar Award (C.S.R.), The Robert A. Welch Foundation (C.S.R.), The National Institutes of Health (C.S.R.), a Beginning Grant in Aid from the American Heart Association (D.-S.L.), and an INSERM Avenir Grant sponsored by La Fondation pour la Recherche Médicale (P.N.).

Author Contributions C.S.R. designed the research. D.-S.L. overexpressed and purified all the proteins used in this work; D.-S.L. and P.N. measured enzyme kinetic data; D.-S.L. generated the crystals; D.-S.L. and C.S.R. collected X-ray diffraction data; P.N. and C.S.R. solved the structures; P.N. did structure refinements; C.S.R. carried out bioinformatic and phylogenetic analyses; M.H. performed GC–MS and radio-HPLC measurements, and determined the structures of the reaction products; and C.S.R. wrote the paper. All authors discussed the results and commented on the manuscript.

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The file contains Supplementary Figures 1- 34 with Legends, Supplementary Tables 1-6, Supplementary Discussion and additional references. (PDF 7022 kb)

Supplementary Movie 1

The file contains Supplementary Movie 1. This movie reveals how the substrate is recognized at the active site of Arabidopsis thaliana allene oxide synthase (At-AOS) (SWF 9060 kb)

Supplementary Movie 2

The file contains Supplementary Movie 2. This movie shows the mode of interaction between the reaction intermediate analog and At-AOS. (SWF 8652 kb)

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Lee, DS., Nioche, P., Hamberg, M. et al. Structural insights into the evolutionary paths of oxylipin biosynthetic enzymes. Nature 455, 363–368 (2008). https://doi.org/10.1038/nature07307

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