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.

18Natural lachrymatory effects are invoked by small volatile S-oxide 19 compounds. They are produced through alkene sulfenic acids by the action of 20 lachrymatory factor synthase (LFS). Here we present the crystal structures of 21 onion LFS (AcLFS) revealed in solute-free and two solute-stabilized forms. 22 Significance statement 38Crushing of onion liberates a volatile compound, syn-propanethial S-oxide 39 (PTSO), which causes lachrymatory effect on humans. We present the crystal 40 structures of onion LFS (AcLFS), the enzyme responsible for natural production 41 of PTSO. AcLFS features a barrel-like fold, and mutagenic and inhibitory 42 analyses revealed that the key residues are present in the central pocket, 43 harboring highly concentrated aromatic residues plus a dyad motif. The 44 architecture of AcLFS is widespread among proteins with various biological 45 functions, such as abscisic acid receptors and polyketide cyclases, and 46 comparisons with these homologs indicate that unique steric and electronic 47 properties maintain the pocket as a reaction compartment. We propose the 48 molecular mechanism behind PTSO generation and shed light on biological 49 decomposition of short-lived sulfur species. 50 51 Results 98Overall structure and confirmation of the active site 99

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