PMC

Lachrymatory sulfine isolated from P. alliacea.

The difference in the mode of action of the onion LFS and P. alliacea LFS.

Alliinase-mediated formation of thiosulfinates from Cys sulfoxide precursors (; ). Alliin is S-allyl-l-Cys sulfoxide, isoalliin is (E)-S-(1-propenyl)-l-Cys sulfoxide, methiin is S-methyl-l-Cys sulfoxide, and propiin is S-propyl-l-Cys sulfoxide.

Cys sulfoxides and their corresponding thiosulfinate derivatives isolated from the Amazonian medicinal plant P. alliacea. The breakdown of the Cys sulfoxides is mediated by P. alliacea alliinase.

Fate of the sulfenic acid intermediate produced by alliinase-catalyzed breakdown of petiveriin. A, When no LFS is present, no PMTSO is formed from PMSA. B, When LFS is present, and depending on its concentration relative to that of the alliinase, the PMSA that is formed can be intercepted by the LFS and converted to PMTSO.

Mechanism advanced by to account for formation of the onion lachrymator, PTSO. Alliinase-bound PLP forms a Schiff base with bound isoalliin. General base catalysis at the active site yields an α,β-unsaturated sulfenic acid that can undergo a [1,4]-sigmatropic rearrangement to furnish the sulfine.

Native PAGE characterization of P. alliacea LFS. A, Native PAGE molecular mass marker. B, Native PAGE with Coomassie Brilliant Blue staining showing the LFS enzyme purified to homogeneity. C, Native PAGE of the LFS after oxidation of carbohydrates bound to the protein, followed by treatment with a hydrazide dye to yield a highly fluorescent conjugate (i.e. a positive test for the presence of sugars).

Confirmation of the production of PMTSO and benzaldehyde by P. alliacea LFS action on the PMSA generated through P. alliacea alliinase-catalyzed breakdown of petiveriin using RP C-18 HPLC. A 10 μL aliquot of a reaction mixture comprised of 1.0 mL of 10 mm phosphate buffer at pH 8.0, 25 μm PLP, 1.5 mm petiveriin, and P. alliacea-derived protein was, after incubation for 10 min at room temperature, analyzed by HPLC (flow rate: 1.0 mL min⁻¹; mobile phase: water:acetonitrile [30:70, v/v]; detection wavelength: 210 nm).

Substrate determination for P. alliacea LFS using RP C-18 HPLC. A 20 μL reaction mixture comprised of 1.0 mL of 10 mm phosphate buffer, pH 8.0, 25 μm PLP, 1.5 mm petiveriin (or 0.25 mm petivericin), and 5.7 μg of purified LFS (approximately 100 nm) at several LFS-to-alliinase molar ratios was incubated for 10 min at room temperature and then analyzed by HPLC to reveal what products, if any, were formed. The substrate used in the reaction for A, B, D, E, and F was petiveriin, and the substrate for C was petivericin. A, Only alliinase was present in the reaction; B and C, only LFS was present in the reaction; D, both LFS and alliinase were present in the reaction, and LFS:alliinase molar ratio was 5:1; E, LFS:alliinase molar ratio was 5:2; F, LFS:alliinase molar ratio was 5:3.

SDS-PAGE analysis of P. alliacea LFS and P. alliacea alliinase. A, Molecular mass marker. B, SDS-PAGE of P. alliacea LFS in the absence of BME, with Coomassie Brilliant Blue staining showing the three bands representing the subunits of which P. alliacea LFS is comprised. C, SDS-PAGE of P. alliacea LFS in the absence of BME after oxidation of carbohydrates bound to the protein, followed by treatment with a hydrazide dye to yield a highly fluorescent conjugate. The results show that the α′-subunit is glycosylated. D, SDS-PAGE of P. alliacea LFS in the presence of BME showing that the α′ band in B collapses into the α band seen in D. E, SDS-PAGE of P. alliacea LFS in the presence of BME after oxidation of carbohydrates bound to the protein, followed by treatment with a hydrazide dye to yield a highly fluorescent conjugate. The results show that the α-subunits are glycosylated. F, SDS-PAGE of P. alliacea alliinase in the absence of BME showing the bands representing the subunits of which P. alliacea alliinase is comprised. G, SDS-PAGE of P. alliacea alliinase in the presence of BME showing that the α′ band in F collapses into the α band. Comparison of D and G reveals that the P. alliacea LFS shares with the P. alliacea alliinase subunits α, γ, and δ, but not its β-subunit. H, SDS-PAGE of P. alliacea alliinase in the presence of BME after oxidation of carbohydrates bound to the protein, followed by treatment with a hydrazide dye to yield a highly fluorescent conjugate. The results show that its α- and β-subunits are glycosylated.