Lachrymatory factor synthase (LFS) is responsible for the natural production of syn-propanethial S-oxide. Because its substrate and product are both transient, the mechanistic insight is currently poor. Here, we decipher the catalytic cycle of onion LFS (AcLFS) by employing crystallography, biochemical assays, and molecular simulations. Crystal structures complexed with solute compounds demonstrate not only the properties of the catalytic site but also a potent binding mode of the labile substrate, a mode which can fulfill the "syn-effect" of the reaction. Mutagenesis analyses identified the key residues in the active site, and the kinetic and energetic backgrounds were specified with computational approaches. We present a rational catalytic mechanism based on the intramolecular proton shuttling that is distinct from the canonical [1,4]-sigmatropic rearrangement reaction. Our investigations both in vitro and in silico provide the mechanistic basis explaining how AcLFS generates the lachrymatory agent in nature and provide insights into the molecular machinery concerning organic labile sulfur species.

Two novel bisthiolane polysulfides (compounds 1 and 2), trivially named thiolanotrisulfide and thiolanotetrasulfide, were isolated from a reaction model of tearless onion (in which lachrymatory factor synthase is suppressed), and the presence of another novel bisthiolane polysulfide (3), trivially named thiolanopentasulfide, was confirmed. On the basis of spectroscopic and mass spectrometric analyses, it was found that these bisthiolane polysulfides were bis(5-hydroxy-3,4-dimethylthiolan-2-yl)-tri/tetra/pentasulfide with the general formulas of C12H22O2S5 (tri-), C12H22O2S6 (tetra-) and C12H22O2S7 (penta-), and they were confirmed to exist in authentic tearless onion juice. Thiolanotrisulfide (1) and thiolanotetrasulfide (2) inhibited cyclooxygenase-1 activity with IC50 values of 720 +/- 78 and 464 +/- 48 mu M respectively, compared with 3282 +/- 188 mu M for aspirin.

Natural lachrymatory effects are invoked by small volatile S -oxide compounds. They are produced through alkene sulfenic acids by the action of lachrymatory factor synthase (LFS). Here we present the crystal structures of onion LFS ( Ac LFS) revealed in solute-free and two solute-stabilized forms. Each structure adopts a single seven-stranded helix-grip fold possessing an internal pocket. Mutagenesis analysis localized the active site to a layer near the bottom of the pocket, which is adjacent to the deduced key residues Arg71, Glu88, and Tyr114. Solute molecules visible on the active site have suggested that Ac LFS accepts various small alcohol compounds as well as its natural substrate, and they inhibit this substrate according to their chemistry. Structural homologs have been found in the SRPBCC superfamily, and comparison of the active sites has demonstrated that the electrostatic potential unique to Ac LFS could work in capturing the substrate in its specific state. Finally, we propose a rational catalytic mechanism based on intramolecular proton shuttling in which the microenvironment of Ac LFS can bypass the canonical [1,4]-sigmatropic rearrangement principle established by microwave studies. Beyond revealing how Ac LFS generates the lachrymatory compound, this study provides insights into the molecular machinery dealing with highly labile organosulfur species. Significance statement Crushing of onion liberates a volatile compound, syn -propanethial S -oxide (PTSO), which causes lachrymatory effect on humans. We present the crystal structures of onion LFS ( Ac LFS), the enzyme responsible for natural production of PTSO. Ac LFS features a barrel-like fold, and mutagenic and inhibitory analyses revealed that the key residues are present in the central pocket, harboring highly concentrated aromatic residues plus a dyad motif. The architecture of Ac LFS is widespread among proteins with various biological functions, such as abscisic acid receptors and polyketide cyclases, and comparisons with these homologs indicate that unique steric and electronic properties maintain the pocket as a reaction compartment. We propose the molecular mechanism behind PTSO generation and shed light on biological decomposition of short-lived sulfur species.

The onion lachrymatory factor (LF) is produced from trans-S-1-propenyl-L-cysteine sulfoxide (PRENCSO) through successive reactions catalyzed by alliinase (EC 4.4.1.4) and lachrymatory factor synthase (LFS), and is responsible for the tear inducing-property and the pungency of fresh onions. We developed tearless, non-pungent onions non-transgenically by irradiating seeds with neon-ion at 20 Gy. The bulbs obtained from the irradiated seeds and their offspring bulbs produced by selfing were screened by organoleptic assessment of tear-inducing property or HPLC analysis of LF production. After repeated screening and seed production by selfing, two tearless, non-pungent bulbs were identified in the third generation (M3) bulbs. Twenty M4 bulbs obtained from each of them showed no tear-inducing property or pungency when evaluated by 20 sensory panelists. The LF production levels in these bulbs were approximately 7.5-fold lower than those of the normal onion. The low LF production levels were due to reduction in alliinase activity, which was a result of low alliinase mRNA expression (less than 1% of that in the normal onion) and consequent low amounts of the alliinase protein. These tearless, non-pungent onions should be welcomed by all who tear while chopping onions and those who work in facilities where fresh onions are processed.

trans-S-1-Propenyl-l-cysteine sulfoxide (PRENCSO) is the most abundant flavor precursor in onions. The changes in PRENCSO, its biosynthetic precursors, and cycloalliin contents in bulbs stored at 0 °C [i.e., recommended by the Food and Agriculture Organization of the United Nations (FAO)] are investigated. Ion-exchange high-performance liquid chromatography analysis revealed larger amounts of PRENCSO and cycloalliin, which were stoichiometrically balanced by the decrease in the combined amounts of the three biosynthetic precursors. The molar balance of the PRENCSO biosynthesis process during onion storage was explained well by the changes of these five compounds. High-throughput integrated liquid chromatography-tandem mass spectrometry analysis was employed to analyze multiple samples at 5 and 20 °C. The molar concentration data, converted using linear regression equations, showed a similar molar balance for the PRENCSO biosynthesis process comprising these five compounds during storage at all temperatures tested. Furthermore, the content of cycloalliin significantly increased at the expense of that of PRENCSO during storage at 20 °C.

S-Alk(en)yl-l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-l-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-l-cysteine to nearly exclusively yield (RC SS )-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-l-cysteine to γ-glutamyl-S-allyl-l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored γ-glutamyl-S-allyl-l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.

In “pinking” of onion, E-(+)-S-(1-propenyl)-l-cysteine sulfoxide is first cleaved by alliinase to yield colour developers (CDs), which react with amino acids, such as valine, to form pigment precursors (PPs). The PPs react with naturally occurring carbonyls (NOCs) to form pigments. By inducing a PP from previously isolated cepathiolanes and l-valine, it was confirmed that cepathiolanes constitute at least a part of the CDs. From the PP and formaldehyde as a NOC, two colourless and two pink compounds were derived. The structure of one of the colourless compounds was established as 2-(2-(1-(1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyrrol-2-yl)methyl-3,4-dimethyl-1H-pyrrol-1-yl)-3-methylbutanoic acid. The structures of the other colourless compound and the pink pigments were predicted based on their molecular formula and the MSn spectral data. A trimeric pigment structure was predicted for one of the pink pigments, which was believed to be the first to be reported in the literature. With these, a new reaction scheme for “pinking” of onion is proposed.

Lachrymatory factor synthase (LFS), an enzyme essential for the synthesis of the onion lachrymatory factor (propanethial S-oxide), was identified in 2002. This was the first reported enzyme involved in the production of thioaldehyde S-oxides via an intra-molecular H(+) substitution reaction, and we therefore attempted to identify the catalytic amino acid residues of LFS as the first step in elucidating the unique catalytic reaction mechanism of this enzyme. A comparison of the LFS cDNA sequences among lachrymatory Allium plants, a deletion analysis and site-directed mutagenesis enabled us to identify two amino acids (Arg71 and Glu88) that were indispensable to the LFS activity. Homology modeling was performed for LFS/23-169 on the basis of the template structure of a pyrabactin resistance 1-like protein (PYL) which had been selected from a BLASTP search on SWISS-MODEL against LFS/23-169. We identified in the modeled structure of LFS a pocket corresponding to the ligand-binding site in PYL, and Arg71 and Glu88 were located in this pocket.

Eight Japanese bunching onion (Allium fistulosum L.) - shallot (Allium cepa L. Aggregatum group) monosomic addition lines (MALs, FF+1A - FF+8A) were used to reveal the effects of single alien chromosomes of A. cepa on the production of amino acids and S-alk(en) yl-L-cysteine sulfoxides (ACSOs) in leaf tissues of A. fistulosum. Determination of amino acid and ACSO content in MAL leaf blades was carried out once every 3 months from August 2005 to May 2006. The amino acid found in the greatest amount in all of the MALs throughout the year was cysteine, except for FF+7A accumulated glycine as the greatest amount of amino acid (November 2005). All of the MALs contained three kinds of ACSOs in varying amounts and proportions, and all accumulated S-1-propenyl CSO as a major ACSO but hardly produced S-2-propenyl CSO throughout the year. FF+3A showed greatly increased proportions of S-methyl CSO in total ACSO, suggesting that anonymous genes controlling S-methyl CSO production are located on chromosome 3A of shallot. High accumulations of total ACSOs in the monosomic additions FF+3A, FF+4A, FF+5A, and FF+8A were observed during different growth periods. Using PCR-based marker analysis, sulfate transporter, adenosine 5'-phosphosulfate reductase (APSR), serine acetyltransferase, O-acetylserine thiol-lyase, glutamylcysteine synthase, glutathione synthase, and gamma-glutamyl transpeptidase candidate genes related to sulfur assimilation and ACSO biosynthesis were allocated to chromosomes 7A, 2A, 7A, 7A, 2A, 7A, and 4A, respectively. This result showed little association between the chromosomal locations of these genes and ACSO accumulation, suggesting that anonymous genes controlling ACSO accumulation were dispersed on the 2A, 3A, 4A, 5A, 7A, and 8A chromosomes of shallot. APSR gene expression was inhibited by 5A chromosome additions, suggesting that one of the regulatory genes was located on a 5A chromosome and inhibited APSR expression.

In normal onion (Allium cepa), trans-S-1-propenyl-L-cysteine sulfoxide (PRENCSO) is transformed via 1-propenesulfenic acid into propanethial S-oxide, a lachrymatory factor (LF), through successive reactions catalyzed by alliinase and lachrymatory factor synthase (LFS). A recent report showed that suppression of the LFS activity caused a dramatic increase in thiosulfinates previously reported as "zwiebelane isomers". After purification by recycle-HPLC and subsequent analyses, we established the planar structure of the putative "zwiebelane isomers" as S-3,4-dimethyl-5-hydroxythiolane-2-yl 1-propenethiosulfinate, in which two of the three molecules of 1-propenesulfenic acid involved in the formation gave the thiolane backbone, and the third molecule gave the thiosulfinate structure. Of at least three stereoisomers observed, one in (2'R,3'R,4'R,5'R)-configuration was collected as an isolated fraction, and the other isomers were collected as a combined fraction because spontaneous tautomerization prevented further purification. Both fractions showed inhibitory activities against cyclooxygenase-1 (COX-1) and alpha-glucosidase in vitro.

We produced a single deuterated lachrymatory factor (propanthial S-oxide, m/z = 91) in a model reaction system comprising purified alliinase, lachrymatory factor synthase (LFS), and (E)-(+)-S-(1-propenyl)-L-cysteine sulfoxide ((E)-PRENCSO) in D(2)O. Onion LFS reacted with the degraded products of (E)-PRENCSO by alliinase, but not with those of (Z)-PRENCSO. These findings indicate that onion LFS is an (E)-1-propenylsulfenic acid isomerase.

In normal onion (Allium cepa), trans-S-1-propenyl-L-cysteine sulfoxide is transformed via 1-propenesulfenic acid into propanethial S-oxide, a lachrymatory factor, through successive reactions catalyzed by alliinase and lachrymatory factor synthase (LFS). A recent report showed that suppression of the LFS activity caused a dramatic increase in thiosulfinates previously reported as "zwiebelane isomers". After purification by recycle high-performance liquid chromatography and subsequent analyses, we established the planar structure of the putative "zwiebelane isomers" as S-3,4-dimethyl-5-hydroxythiolane-2-yl 1-propenethiosulfinate, in which two of the three molecules of 1-propenesulfenic acid involved in the formation gave the thiolane backbone, and the third molecule gave the thiosulfinate structure. Of at least three stereoisomers observed, one in the (2'R,3'R,4'R,5'R)-configuration was collected as an isolated fraction, and the other isomers were collected as a combined fraction because spontaneous tautomerization prevented further purification. Both fractions showed inhibitory activities against cyclooxygenase-1 and α-glucosidase in vitro.

Eight Japanese bunching onion (Allium fistulosum L.)—shallot (Allium cepa L. Aggregatum group) monosomic addition lines (MALs, FF + 1A–FF + 8A) were used to reveal the effects of single alien chromosomes of A. cepa on the production of amino acids and S-alk(en)yl-L-cysteine sulfoxides (ACSOs) in leaf tissues of A. fistulosum. Amino acid and ACSO contents in MAL leaf blades were determined once every 3 months from August 2005 to May 2006. The amino acid found in the greatest amount in all of the MALs throughout the year was cysteine, except for FF + 7A accumulated glycine as the greatest amount of amino acid (Nov. 2005). All of the MALs contained three ACSOs in varying amounts and proportions, and all accumulated S-1-propenyl CSO as a major ACSO but hardly produced S-2-propenyl CSO throughout the year. FF + 3A showed greatly increased proportions of S-methyl CSO in total ACSO, suggesting that anonymous genes controlling S-methyl CSO production are located on chromosome 3A of shallot. High accumulation of total ACSOs in the monosomic additions FF + 3A, FF + 4A, FF + 5A, and FF + 8A was observed during different growth periods. Using PCR-based marker analysis, sulfate transporter, adenosine 5'-phosphosulfate reductase (APSR), serine acetyltransferase, O-acetylserine thiol-lyase, glutamylcysteine synthase, glutathione synthase, and γ-glutamyl transpeptidase candidate genes related to sulfur assimilation and ACSO biosynthesis were allocated to chromosomes 7A, 2A, 7A, 7A, 2A, 7A, and 4A, respectively. This result showed little association between the chromosomal locations of these genes and ACSO accumulation, suggesting that anonymous genes controlling ACSO accumulation were dispersed on the 2A, 3A, 4A, 5A, 7A, and 8A chromosomes of shallot. APSR gene expression was inhibited by 5A chromosome additions, suggesting that one of the regulatory genes was located on a 5A chromosome and inhibited APSR expression.

To use synthesized onion lachrymatory factor (SOLF) to investigate age-related changes in reflex-tear secretion and ocular-surface sensation.We separated 91 healthy volunteers into four groups: groups A, age 20-29 years; B, 30-39; C, 40-49; and D, older than 50 years. We exposed one eye of each subject to SOLF and measured the elapsed time until the subject's limit of irritation tolerance (TLI) was reached and an increase in the tear meniscus radius (DeltaR). After the SOLF stimulus, corneal sensitivity was examined by Cochet-Bonnet esthesiometry (CB), and reflex-tear secretion was examined by the Schirmer I-test (ST).TLI was significantly shorter in group A than in the other groups (P < 0.0001), and the groups B and D also differed significantly from each other (P = 0.0013). The increase in DeltaR was significantly greater in group A than in group C (P = 0.0306) or D (P < 0.0001), and groups B (P = 0.0002) and C (P = 0.0308) also differed significantly from group D. There were no significant intergroup differences in the CB and ST results.An age-related decrease in reflex-tear secretion and ocular-surface sensation was revealed by the SOLF test but could not be detected by either CB or the ST.