Biosci. Biotechnol. Biochem., 76 (9), 1799–1801, 2012 Note Proton Transfer in a Reaction Catalyzed by Lachrymatory Factor Synthase

y Noriya MASAMURA, Morihiro AOYAGI, Nobuaki TSUGE, Takahiro KAMOI, and Shinsuke IMAI

Somatech Center, House Foods Corporation, Takanodai, Yotsukaido, Chiba 284-0033, Japan

Received May 1, 2012; Accepted June 11, 2012; Online Publication, September 7, 2012 [doi:10.1271/bbb.120338]

We produced a single deuterated lachrymatory factor Garlic alliinase was purified by the method of Nock et (propanthial S-oxide, m=z ¼ 91) in a model reaction al.11) Alliinase activity was quantified by pyruvate system comprising purified alliinase, lachrymatory formation based on a coupling assay involving the factor synthase (LFS), and (E)-(þ)-S-(1-propenyl)-L- oxidation of NADH to NAD by lactate dehydrogenase, 12) ((E)-PRENCSO) in D2O. Onion LFS as previously described by Imai et al. A single unit of reacted with the degraded products of (E)-PRENCSO activity was defined as that capable of producing 1 mmol by alliinase, but not with those of (Z)-PRENCSO. These pyruvate from (E)-PRENCSO in 1 min at room temper- findings indicate that onion LFS is an (E)-1-propenyl- ature. Recombinant onion LFS protein was prepared . following published protocols.13) (E)-PRENCSO was purified from onion bulbs following the protocol Key words: Allium cepa; lachrymatory factor synthase; described by Shen and Parkin.7) PRENCSO was synthe- þ 14) intra-molecular H substitution; D2O; sub- sized by the method of Lancaster and Kelly, and the strate specificity (E)- and (Z)-forms were separated by reversed-phase HPLC: Pegasil ODS (20 mm 250 mm), pH 3.3 H2O Although have significant nutritional and (TFA), 5 mL/min, 230 nm. organoleptic values, it is not uncommon to experience A small amount (2.5 mg) of (E)-PRENCSO was a ‘‘crying’’ sensation when cutting and chopping onion resolved with 1 mL D2O (151882, 99.9 atom % bulbs. The substance that causes this irritation is called deuterium, Sigma-Aldrich, St. Louis, MO) or deionized the lachrymatory factor (LF), and was identified as H2O. Paper pads were blotted with garlic alliinase (7.59 propanthial S-oxide by Brodnitz and Pascale more than units) and excess onion LFS, and lyophilized. The 35 years ago.1) The LF is a rare compound belonging to resulting lyophilized paper pads were placed on the the group of thioaldehyde S-oxides. To date, only three bottom of a 20 mL gas chromatography (GC) headspace other naturally occurring thioaldehyde S-oxides have vial, and 90 mL of the PRENCSO solution was added. been reported.2–4) The vial was immediately closed with a lid, and was Onion LF was widely believed to be non-enzymati- maintained at room temperature for approximately cally produced from (E)-1-propenylsulfenic acid, a 5 min. The headspace of the vial was applied to the putative reaction of alliinase and (E)-(þ)-S-(1- 5975 MSD MS system (Agilent Technologies, Santa propenyl)-L-cysteine sulfoxide ((E)-PRENCSO)), which Clara, CA) equipped with a 7890A GC system (Agilent is abundant in the onion.5–8) In opposition to this, we Technologies, Santa Clara, CA). GC separation was discovered lachrymatory factor synthase (LFS), an achieved using a 60 m 250 mm 0:25 mm column essential in the formation of the LF after (J&W 122–7062, Agilent Technologies, Santa Clara, alliinase acts on (E)-PRENCSO, and cloned its cDNA CA). The carrier gas was helium at a flow of 1.2 mL/ from an onion bulb (Allium cepa).9) min, and the column temperature program included an Based on chemical analysis of freshly prepared onions increase of 10 C/min from 40 C to 210 C. The crushed in D2O, it has been speculated that the LFS detector temperature was held constant at 230 C, and catalyzes the intra-molecular Hþ substitution reaction total ion chromatograms and mass spectra were analyzed between atom 1 (oxygen) and atom 4 (carbon) in (E)-1- in the electron-impact mode. Selected ion monitoring propenylsulfenic acid (Fig. 1),10) but that study did not (SIM) was used to identify the incorporation of exclude the possibility that other unknown reactions are deuterium. involved in LF formation. Moreover, the detailed As shown in Fig. 2, when LF formation proceeded in mechanism of this enzyme has not yet been published, D2O, an m=z peak of 91 was detected (Fig. 2b, c). This probably due to (E)-1-propenylsulfenic acid instability. result strongly suggests that the exchangeable proton In this study, we reacted purified garlic alliinase, connecting atom 1 (oxygen) in (E)-1-propenylsulfenic onion LFS, and (E)-PRENCSO in D2O to show that a acid was replaced with deuterium, resulting in the proton transfer occurs during the formation of onion LF. production of a single deuterated LF (LF-d, m=z ¼ 91). The selectivity of the LFS for (E)- and (Z)- Thus LFS catalyzed the isomerization of sulfenic acid to PRENCSO was also examined to provide corroborative LF. It is also suggested that the deuterium was evidence as to the reaction mechanism. incorporated into the alkyl chain (CH3–CH2–CH=) of

y To whom correspondence should be addressed. Tel: +81-43-237-5211; Fax: +81-43-237-2914; E-mail: [email protected] Abbreviations: LF, lachrymatory factor; LFS, lachrymatory factor synthase; (E)-PRENCSO, (E)-(þ)-S-(1-propenyl)-L-cysteine sulfoxide 1800 N. MASAMURA et al.

Fig. 1. Proposed Reaction Pathway from (E)-1-Propenylsulfenic Acid to LF.

30,000 a

20,000 propanal LF Abundance

10,000

2,000 7.008.00 9.00 10.00 11.00 12.00 13.0014.00 15.00 min

4,000 b 3,000 m/z = 90

2,000 Abundance

1,000

0 7.008.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 min

4,000 c m/z = 91 3,000

2,000 Abundance

1,000

0 7.008.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 min

91.0

4,000 d

3,000

2,000 Abundance

74.0 1,000 48.0

58.0 62.9 70.8 54.0 67.9 77.0 87.0 101.9 120.9 0 m/z

Fig. 2. GS-MS Analysis of the Head Space of the Reaction Mixture. Analysis of the reaction mixture in D2O by total ion chromatography (a) and selected ion monitoring (SIM) of m=z of 90 (b) and 91 (c). Mass spectra of the peak detected in the SIM of m=z of 91 (d). Reaction Mechanism of Onion LFS 1801

- O NH which it can react, and confirms our speculation as to 80 2 + the position of deuterium in LF-d. The most feasible S 60 COOH explanation as to why the isomeric conversion from (Z)- (E) -PRENCSO propenylsulfenic acid to LF did not proceed is that the 40 hydrogen connecting with atom 4 (carbon) was located far from the exchangeable proton connecting with atom 20 LF peak area (mV * s) - 1 (oxygen) in (Z)-propenylsulfenic acid. O NH2 0 + Hence we propose that onion LFS should be classified S COOH as an (E)-1-propenylsulfenic acid isomerase. (Z) -PRENCSO -PRENCSO -PRENCSO -isomer References (E) (E) (Z) Natural PRENCSO 1) Brodnitz MH and Pascale JV, J. Agric. Food Chem., 19, 269– 272 (1971). Fig. 3. Substrate Selectivity of Onion LFS. 2) Block E and Bayer T, J. Am. Chem. Soc., 112, 4584–4585 LF-forming activity is indicated as LF peak area following (1990). 13) Masamura et al. 3) Kubec R, Kim S, and Musah RA, Phytochemistry, 63, 37–40 (2003). 4) Kubec R, Cody RB, Dane AJ, Musah RA, Schraml J, LF-d, because the m=z ¼ 48 fragment, which was Vattekkatte A, and Block E, J. Agric. Food Chem., 58, 1121– derived from the S=O moiety of LF-d, was detected 1128 (2010). in the mass spectra of the LF-d peak (Fig. 2d). Although 5) Block E, Penn RE, and Revelle LK, J. Am. Chem. Soc., 101, it is difficult to determine which hydrogen in the alkyl 2200–2201 (1979). chain was replaced by deuterium, Block et al. (1996) 6) Block E, Angew. Chem. Int. Ed. Engl., 31, 1135–1178 (1992). reported that the hydrogen connecting atom 4 (carbon) 7) Shen C and Parkin KL, J. Agric. Food Chem., 48, 6254–6260 (2000). was selectivity deuterated in a pyrolysis experiment in 8) Yamane A, Yamane A, and Shibamoto T, J. Agric. Food Chem., 10) D2O. Hence we speculate that the hydrogen at atom 4 42, 1010–1012 (1994). (carbon) is exchanged with the deuterium in LF-d. 9) Imai S, Tsuge N, Tomotake M, Nagatome Y, Sawada H, Nagata In addition, a high degree of deuterium enrichment T, and Kumagai H, Nature, 419, 685 (2002). (87%) was observed. Deuterium-labeled LF might help 10) Block E, Gillies JZ, Gillies CW, Bazzi AA, Putman D, Revelle further understand the mechanism of thiosulfinate for- LK, Wang D, and Zhang X, J. Am. Chem. Soc., 118, 7492–7501 mation following LF formation. In the D O reaction, (1996). 2 11) Nock LP and Mazelis M, Phytochemistry, 28, 729–731 (1989). deuterium-labeled propanal was detected (Fig. 2a). 12) Imai S, Akita K, Tomotake M, and Sawada H, J. Agric. Food Although the pathway of desulfuration is unclear, Chem., 54, 848–852 (2006). propanal is a known decomposition product of the LF.15) 13) Masamura N, Ohashi W, Tsuge N, Imai S, Ishii-Nakamura A, The amount of LF produced by each of the substrates, Hirota H, Nagata T, and Kumagai H, Biosci. Biotechnol. (E)-PRENCSO and (Z)-PRENCSO, was directly com- Biochem., 76, 447–453 (2012). pared. Although alliinase has been reported to hydrolyze 14) Lancaster JE and Kelly KE, J. Sci. Food Agric., 34, 1229–1235 both PRENCSOs equally,16) the degraded products of (1983). 15) Block E, ‘‘Garlic and Other Alliums the Lore and the Science,’’ (Z)-PRENCSO by alliinase did not lead to LF formation RSC publishing, Cambridge, pp. 107–132 (2010). (Fig. 3). This indicates that the LFS has a high 16) Freeman GG and Whenham RJ, J. Sci. Food Agric., 26, 1333– selectivity for (E)-propenylsulfenic acid, a putative 1346 (1975). reaction product derived from (E)-PRENCSO, with