Created by: Hannah Chung

Lachrymatory factor synthase (PDB ID: 5VGS, LFS) is an isomerase enzyme found in Allium cepa that converts (E)-1-propenesulfenic acid to (Z)-propanethial S-oxide, the lachrymatory factor (LF) (1). LFS is an isomerase because of the rearrangement reaction that converts the substrate to the (Z)-phenylmethanethial S-oxide isomer without changing the chemical formula (1). In a proposed enzymatic mechanism, LFS facilitates an intramolecular proton exchange between the oxygen and the alkene chain of its substrate (1). The volatile LF is notorious for causing eye irritation as part of the chemical defense mechanism against microbes and animals (1). The Allium species are known for health benefits such as anti-inflammation, anticancer, lipid-lowering, and antiplatelet aggregation (2). Suppression of LFS expression resulted in a “tearless” onion that did not trigger eye irritation upon rupture of the onion tissue (1). The production of LF is hypothesized to restrict the production of other sulfur volatiles such as those native to garlic (2).

The catalytic activity of LFS depends on its structure. The molecular weight and isoelectric point of LFS were calculated with ExPASy to be 19056.73 daltons and 5.02, respectively (3). LFS is a monomer with one chain of 157 amino acids and the secondary structure is composed of 25% α-helices and 43% β-sheets (4). LFS shows a compact fold made up of seven strands of antiparallel β-sheets (strands β1— β7) that encircle a long C-terminal α-helix with two additional shorter helices between the first two β-sheets (1). The tertiary structure resembles the helix-grip fold conformation (1). The dominant feature of LFS is the large internal hydrophobic cavity of an elongated shape, formed by the inside surface of curved β-sheets and α-helices (1). The volume of the binding pocket is reduced to approximately 216 ų because of the extended β3—β4 loop and two tryptophan residues, Trp-133 and Trp-155 (1). The reduced size of the cavity in LFS is accompanied by a substitution of the lysine and the cluster of polar amino acids with methionine, phenylalanine, leucine, and tryptophan hydrophobic residues (1). The reduced binding pocket size and elevated hydrophobicity demonstrate the primary adaptation of LFS for specific interaction with the small 1-propenesulfenic acid substrate.

The intramolecular cavity in LFS is hypothesized to be the active site of the enzyme (1). Researchers had difficulties in obtaining insight into the specific mode of substrate—enzyme interaction because of the high propensity for self-condensation of LFS (1).  However, there is a nonpolar cap at the putative entrance of the binding site to shield the cavity from the environment (1). The enfolded side chains are composed of two methionine residues, Met-77 and Met-143 from the β3—β4 and β7—α3 loops, and a phenylalanine residue, Phe-108 from the β5—β6 loop (1). It is probable that the relative positions of these side chains depend on the orientation of relatively flexible portal loops to allow the spontaneous diffusion of the substrate in and out of the active site (1). The other side of the binding cavity is covered primarily by the polar side chains of α2 and α3 that also form a narrow tunnel leading to the active site (1). In the crystal structure of LFS, the tunnel is occupied by highly ordered water molecules, connecting the exterior of the enzyme to the active site (1).

Crotyl alcohol of both configurations was used in experimentation to map the active site of LFS because of its structural similarity to (E)-1-propenesulfenic acid (1). Based on the data, there are two distinct modes of crotyl alcohol binding that are dependent on the configuration of the ligand (1). The (Z)-isomer has a hydrogen bond between the hydroxyl group of the ligand and the side chains of two tyrosine residues, Tyr-102 and Tyr-124 (1). The hydrophobic chain of the (Z)-isomer projected toward the nonpolar portion of the binding pocket formed by Leu-47, Val-73, Phe-84, and Trp-133 (1). The (E)-isomer of crotyl alcohol adopted an alternate orientation with the hydroxyl group facing the polar side chains of Glu-88 and Arg-71 residues while also remaining within 3.3 Å proximity to the β carboxyl group of Glu-88 (1). The orientation of this isomer was defined by the hydrophobic interaction of the alkene chain with the side chains of Phe-84, Phe-148, Tyr-102, Trp-133, Trp-155, and Leu-152 residues (1). Although the chemical differences between crotyl alcohol and (E)-1-propenesulfenic acid may lead to alternate binding for the substrate, crotyl alcohol provides crucial insight in the mechanism of binding (1). 

A substrate-docking experiment using AutoDock Vina assessed potential differences between the interaction of LFS with crotyl alcohol and (E)-1-propenesulfenic acid (1). The spatial orientation of the bound (E)-1-propenesulfenic acid differed from that observed with crotyl alcohol (1). The preferred orientation of (E)-1-propenesulfenic acid engaged the alkene chain in hydrophobic interactions with Trp-133, Trp-155, Phe-148, and Leu-152 residues (1). The oxygen atom of the substrate participated in hydrogen bonding with the carbonyl group of the Glu-88 side chain, the hydroxyl portion of Tyr-102, and the amino group of Arg-71 (1). The substrate adopted a synperiplanar orientation, consistent with the final Z configuration of the final product (1). This implied that the substrate binds to the active site in a preferential configuration to produce only (Z)-phenylmethanethial S-oxide (1).

The two solvent-inaccessible polar amino acids, Glu-88 and Arg-71, were found in close proximity to the substrate molecule and are crucial to LF formation (1). Glu-88 is hypothesized to polarize the substrate by removing a proton in a general base mechanism (1). The neighboring Arg-71 stabilizes the negative charge on Glu-88 through hydrogen bonding to prevent extraneous protonation under cellular conditions (1). After proton extraction from the substrate, a transitory double bond forms between the oxygen and sulfur atoms that immediately rearranges to form S-oxide (1). The hydroxyl group of the Tyr-102 residue or another proton donating residue may stabilize the carbocation intermediate that forms by providing a new hydrogen atom bond to yield the final product (1). This mechanism to form LF is not found in other plant species and is formally a rearrangement reaction (1).  

A comparison search for similarities in sequence and structure conducted through the Dali Server and the Protein- Specific Iterated Basic Local Assignment Search Tool (PSI-BLAST) searches yielded abscisic acid (ABA) receptor pyrabactin resistance (PDB ID: 3K90, PYR1) from Arabidopsis thaliana as a suitable comparison protein. The Dali Server compiles a list of proteins with similar tertiary structures by using a sum-of-pairs method that calculates differences in intramolecular distances, assigning a Z-score to each protein (5). A Z-score greater than 2 indicates high similarity (5). PSI-BLAST is a program that searches for proteins with similar primary structures and assigns an E-score based on gaps in the sequence (6). An E-score below 0.05 indicates high similarity between proteins (6). The ABA receptor received a Z-score of 16.7 and an E-score of 2e-5, which implies noteworthy structural and sequence similarity between the ABA receptor and LFS (5,6).

The secondary structure of PYR1 is composed of 25% α-helices and 31% β-sheets, similar to that found in LFS (4). LFS and the monomer of PYR1 share similarities in their tertiary structure. PYR1 contains a Bet v I domain which is composed of seven β strands forming a pronounced central cavity much like the hydrophobic binding pocket of LFS (7). Both proteins are structured specifically for each respective substrate. PYR1 almost completely encloses ABA within the cavity and stabilizes the hormone through favorable interactions with the polar and hydrophobic character of the residues (7). The polar residues of the amine group in Lys-59 and the backbone amide of Ala-89 found in the PYR1 cavity stabilize the substrate like those found in the LFS binding pocket (1,7). The nonpolar residues that conform the upper part of the PYR1 cavity are Phe-61, Val-163, and Val-83 that effectively participate in van der Waals interactions to further stabilize the substrate and shield the ligand from the solvent, similar to the residues that form the nonpolar cap of the LFS cavity (1,7).

A key difference between PYR1 and LFS is that PYR1 functions as a dimer while LFS acts only as a monomer (7). The two subunits of PYR1 are very similar but contain specific differences in residues 84-89, 113-118, and 153-159 in the β3—β4 and β5—β6 loops and the N-terminal portion of the α5 helix that surround the upper part of the PYR1 cavity to protect the substrate almost entirely from the solvent in the closed conformation (7). The loops of the subunits are important to stabilize the cavity’s entrance and the ligand within the cavity and demonstrate the role of the PYR1 dimer as an effective hormone receptor (7). The conformation change upon binding to the hormone allows PYR1 to regulate phosphorylation of serine/threonine protein kinases (8). Increase in ABA levels induce the formation of a complex between PYRs and protein phosphatases type 2C (PP2C) that inactive PP2C phosphatase activity, enabling accumulation of active, phosphorylated sucrose non-fermenting1-related subfamily 2 (SnRK2) that are mediators of stress adaptations like stomatal closure (8).

Another noteworthy difference between the two binding pockets is the relative size. The PYR1 cavity is significantly larger than the respective substrate, which occupies the upper part, and leaves room for water molecules and phosphates which suggests that both are able to bind to the ABA hormone (7). The wider cavity also implies that both PYR1 subunits are able to bind to the hormone in solution (7). In contrast to LFS, the cavity is reduced in size to selectively bind only to the small (E)-1-propenesulfenic acid molecule (1).

In terms of function, the hormone ABA plays a key role in signaling for many plant processes such as root growth, stomatal opening, seed maturation and dormancy, and response to abiotic stresses like drought, cold weather, and salinity (7). This contrasts to the function of LFS as an isomerase (1). The observed differences in function may be due to the fact that PYR1 is a two subunit hormone receptor and changes conformation upon binding to the ligand to activate the pathway to plant stress response whereas LFS is an isomerase enzyme that converts (E)-1-propenesulfenic acid to an alternative conformation (1). The PYR1 cavity is specific to its substrate and thus will only change conformation when it binds to the hormone, propagating the signal and the appropriate stress response (7). Similarly, LFS will not alter the conformation of any other substrate besides (E)-1-propenesulfenic acid because of the specificity of the hydrophobic cavity (1). The PYR1 and LFS proteins are both biologically significant to their respective organisms and function through the respective specificity of their hydrophobic cavities.   

In conclusion, LFS is an important isomerase enzyme in A. cepa that converts (E)-1-propenesulfenic acid to the eye irritant LF that protects the organism from animals and microbes (1). LFS carries out its function through the steric and hydrophobic specificity of the binding cavity (1). The mechanism of LFS remains largely unknown but provides a fascinating example of an enzymatic protein containing the hydrophobic binding cavity structure. This represents the first step in understanding the striking eye-irritating property of onions.