title: Crystal structures of Physcomitrella patens AOC1 and AOC2: insights into the enzyme mechanism and differences in substrate specificity.
abstract: In plants, oxylipins regulate developmental processes and defense responses. The first specific step in the biosynthesis of the cyclopentanone class of oxylipins is catalyzed by allene oxide cyclase (AOC) that forms cis(+)-12-oxo-phytodienoic acid. The moss Physcomitrella patens has two AOCs (PpAOC1 and PpAOC2) with different substrate specificities for C₁₈- and C₂₀-derived substrates, respectively. To better understand AOC's catalytic mechanism and to elucidate the structural properties that explain the differences in substrate specificity, we solved and analyzed the crystal structures of 36 monomers of both apo and ligand complexes of PpAOC1 and PpAOC2. From these data, we propose the following intermediates in AOC catalysis: (1) a resting state of the apo enzyme with a closed conformation, (2) a first shallow binding mode, followed by (3) a tight binding of the substrate accompanied by conformational changes in the binding pocket, and (4) initiation of the catalytic cycle by opening of the epoxide ring. As expected, the substrate dihydro analog cis-12,13S-epoxy-9Z,15Z-octadecadienoic acid did not cyclize in the presence of PpAOC1; however, when bound to the enzyme, it underwent isomerization into the corresponding trans-epoxide. By comparing complex structures of the C₁₈ substrate analog with in silico modeling of the C₂₀ substrate analog bound to the enzyme allowed us to identify three major molecular determinants responsible for the different substrate specificities (i.e. larger active site diameter, an elongated cavity of PpAOC2, and two nonidentical residues at the entrance of the active site).
authors.affiliation: Department of Molecular Structural Biology, Institute of Microbiology and Genetics, University of Goettingen, 37077 Goettingen, Germany.
authors.forename: Piotr
authors.initials: P
authors.lastname: Neumann
authors.forename: Florian
authors.initials: F
authors.lastname: Brodhun
authors.forename: Kristin
authors.initials: K
authors.lastname: Sauer
authors.forename: Cornelia
authors.initials: C
authors.lastname: Herrfurth
authors.forename: Mats
authors.initials: M
authors.lastname: Hamberg
authors.forename: Jens
authors.initials: J
authors.lastname: Brinkmann
authors.forename: Julia
authors.initials: J
authors.lastname: Scholz
authors.forename: Achim
authors.initials: A
authors.lastname: Dickmanns
authors.forename: Ivo
authors.initials: I
authors.lastname: Feussner
authors.forename: Ralf
authors.initials: R
authors.lastname: Ficner
mesh_terms: D055162:Biocatalysis; D053898:Biosynthetic Pathways; D019068:Bryopsida / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003517:Cyclopentanes / Q000737:chemistry / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D008958:Models, Molecular; D054883:Oxylipins / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
chemical_list: D003517:Cyclopentanes; D005231:Fatty Acids, Unsaturated; D007527:Isoenzymes; D054883:Oxylipins; C007201:cyclopentanone; C027369:octadecadienoic acid; C011006:jasmonic acid; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase