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.

Cytochrome P450 isolated from Bacillus subtilis (P450 BS␤ ; molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the ␣-and ␤-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450 BS␤ in the substrate-bound form, determined at a resolution of 2.1 Å. P450 BS␤ exhibits a typical P450 fold. The substrate binds to a specific channel in the enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by electrostatic interaction of its terminal carboxylate with the Arg 242 guanidium group. These interactions are responsible for the site specificity of the hydroxylation site in which the ␣-and ␤-positions of the fatty acid come into close proximity to the heme iron sixth site. The fatty acid carboxylate group interacts with Arg 242 in the same fashion as has been reported for the active site of chloroperoxidase, His 105 -Glu 183 , which is an acid-base catalyst in the peroxidation reactions. On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by P450 BS␤ is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.Two bacterial cytochrome P450s isolated from Sphingomonas paucimobilis and Bacillus subtilis, P450 SP␣ 1 and P450 BS␤ , respectively, are heme-containing enzymes that catalyze the hydroxylation reaction of long chain fatty acids (e.g. myristic acid) using hydrogen peroxide (H 2 O 2 ) as an oxidant to produce hydroxylated (-OH) fatty acids (1, 2). In the enzymatic reactions, an oxygen atom derived from H 2 O 2 is efficiently introduced into the substrate with a high catalytic turnover (1,000 min Ϫ1 ) (2-4). P450 SP␣ produces the ␣-OH fatty acid (100%) as the product, whereas P450 BS␤ produces both the ␤-OH (60%) and the ␣-OH (40%) fatty acids (1, 2, 4, 5). The amino acid sequence of the two enzymes shares a 44% identity (2). Data base investigation has shown that P450 SP␣ and P450 BS␤ belong to the P450 superfamily and, therefore, they have been given the systematic nomenclature designations CYP152B1 and CYP152A1, respectively (6). However, when compared with reactions catalyzed by other P450s, two characteristic properties in the P450 SP␣ and P450 BS␤ reactions were found, i.e. the utilization of H 2 O 2 and the site specificity of the reaction.In typical P450 reactions an oxygen atom derived from molecular oxygen (O 2 ) is inserted into the substrates (7), and the reaction is referred to as a monooxygenation reaction. Two protons and two electrons are required in the monooxygenation reaction. The electrons are supplied from NAD(P)H through mediation by flavoproteins and iron-sulfur proteins, and the protons are probably delivered from solvent water to the active site through a specific hydrogen-bonding network (8). In the monooxygenase P450 system, H 2 O 2 is sometimes used as a surrogate for the O 2 /2e Ϫ /2H ϩ system (peroxide sh...

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Several sampling techniques based on steam distillation (SD), simultaneous distillation and solvent extraction (SDE), solid-phase trapping solvent extraction (SPTE), and headspace solid-phase microextraction (HS-SPME) have been compared for the determination of Korean garlic flavor components by gas chromatography-mass spectrometry (GC-MS). Diallyl disulfide (57.88%), allyl sulfide (23.59%), and diallyl trisulfide (11.40%) were found to be the predominant flavor components of garlic samples extracted by SDE whereas these components were at levels of 89.77%, 2.43%, and 3.89% when the same sample was extracted by SD, 97.77%, 0.17%, and 0.10% by SPTE, and 97.85%, 0.01%, and 0.01% by HS-SPME using the 50/30-microm divinyl benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber. Thermal degradation of components such as allyl methyl sulfide, dimethyl disulfide, and thiirane were observed for SDE and SD but not for SPTE or HS-SPME. HS-SPME had several advantages compared with SD, SDE, and SPTE-rapid solvent-free extraction, no apparent thermal degradation, less laborious manipulation, and less sample requirement. Five different fiber coatings were evaluated to select a suitable fiber for HS-SPME of garlic flavor components. DVB/CAR/PDMS was most efficient among the five types of fiber investigated.