Abstract
A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is a heterotetrameric glycoprotein comprised of two alpha-subunits (68.8 kD each), one gamma-subunit (22.5 kD), and one delta-subunit (11.9 kD). The two alpha-subunits are glycosylated and connected by a disulfide bridge. The LFS has an isoelectric point of 5.2. It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-l-cysteine sulfoxide (petiveriin). Depending on its concentration relative to that of P. alliacea alliinase, the LFS sequesters, to varying degrees, the sulfenic acid intermediate formed by alliinase-mediated breakdown of petiveriin. At LFS:alliinase of 5:1, LFS sequesters all of the sulfenic acid formed by alliinase action on petiveriin, and converts it entirely to (Z)-phenylmethanethial S-oxide. However, starting at LFS:alliinase of 5:2, the LFS is unable to sequester all of the sulfenic acid produced by the alliinase, with the result that sulfenic acid that escapes the action of the LFS condenses with loss of water to form S-benzyl phenylmethanethiosulfinate (petivericin). The results show that the LFS and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the LFS acts. The results also show that the LFS modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the LFS and the alliinase. These observations suggest that manipulation of LFS-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules.
Highlights
A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea
Since phenylmethanethial S-oxide (PMTSO) production was only observed when LFS was exposed to both petiveriin and P. alliacea alliinase, the presence of the LFS in protein fractions was determined by tracking which protein fractions, when combined with a solution of petiveriin and P. alliacea alliinase in buffer, reliably produced PMTSO, as monitored by reversed-phase (RP) C-18 HPLC (Fig. 3)
Over the course of these experiments, a chromatographic protocol was developed that resulted in the isolation of purified LFS whose activity in the production of PMTSO could be completely separated from the activity of the P. alliacea alliinase
Summary
A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The LFS has an isoelectric point of 5.2 It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-L-cysteine sulfoxide (petiveriin). The Cys sulfoxide that serves as the precursor of the onion lachrymator is (E)-S-(1-propenyl)-L-Cys sulfoxide (isoalliin) It is structurally distinct from other naturally occurring S-substituted Cys sulfoxides so far reported in that it is a,b-unsaturated. This structural feature affords its corresponding 1-propenylsulfenic acid (PSA) the possibility of undergoing a [1,4]-sigmatropic rearrangement that, in principle, would furnish the onion lachrymator, PTSO. We describe our confirmation of the existence of a LFS in P. alliacea, and detail biochemical characterization of this novel class of enzymes
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