Aged black garlic-derived S-allyl-L-cysteine serves as a dietary precursor for supersulfide production by cystathionine γ-lyase

Abstract

S-allyl-L-cysteine (SAC) is a major bioactive compound in aged black garlic with multiple pharmacological benefits, including antioxidant and neuroprotective effects; however, the underlying molecular mechanisms of these benefits remain unclear. In our previous study, we demonstrated that cystathionine γ-lyase (CSE), a sulfur-containing amino acid-metabolizing enzyme involved in the transsulfuration pathway, metabolizes S-nitroso-L-cysteine via a C-S lyase reaction. In this study, we investigated whether CSE-mediated metabolism is involved in the physiological effects of various cysteine derivatives, including SAC. We evaluated the potential of a series of S-alkylcysteine derivatives with varying alkyl chain lengths and degrees of unsaturation as CSE substrates. Recombinant CSE experiments revealed that thiol compounds were produced from propyl derivatives, whereas supersulfides were generated from ethyl, propyl, and allyl derivatives. Kinetic analysis demonstrated that the allyl derivative exhibited a stronger substrate affinity than the propyl derivative, similar to that of the endogenous substrate cystathionine. This study suggests a novel molecular basis for CSE-mediated metabolism and subsequent supersulfide production, which contributes to the diverse health-promoting effects of SAC in mammalian tissues.

INTRODUCTION

S-alkylcysteines have garnered considerable attention as safe and effective dietary sulfur sources. S-allyl-L-cysteine (SAC) is abundantly present in aged black garlic (Amagase, 2006). Growing evidence indicates that these cysteine derivatives exhibit a remarkably diverse range of health-promoting and pharmacological effects. These beneficial activities are shared among various S-alkyl counterparts: SAC, S-propyl-L-cysteine (SPC), and S-ethyl-L-cysteine (SEC), which possess potent antioxidant, anti-inflammatory, neuroprotective, and lipid-lowering properties (Liu and Yeh, 2002; Tsai et al., 2011; Imai et al., 2016; Khajevand-Khazaei et al., 2019). Despite their broad pharmacological spectra and functional similarities, the core molecular mechanisms underlying the multiple physiological activities of these structurally related compounds remain unclear.

Cystathionine γ-lyase (CSE) is a sulfur-containing amino acid-metabolizing enzyme (Sbodio et al., 2019). Recently, we demonstrated that CSE possesses robust C-S bond-cleaving (α, β-elimination) capabilities and metabolizes S-nitroso-L-cysteine to produce thionitrous acid and pyruvate. Importantly, supersulfides were also generated during this reaction (Araki et al., 2025). These findings raised the possibility that CSE may also metabolize other sulfur-containing cysteine derivatives to generate supersulfides. Supersulfides are a group of sulfur species with catenated sulfur atoms, including hydrogen polysulfides (H₂S_n, n > 1) and organic polysulfides (RSS_nR, n > 1), which exhibit nucleophilic and antioxidant properties that are stronger than those of conventional thiols and function as crucial redox signaling molecules (Akaike et al., 2024; Barayeu et al., 2026). Furthermore, they are closely associated with energy metabolism in the mitochondrial electron transport chain and regulate the mitochondrial lifespan (Akaike et al., 2017). Recent studies have demonstrated that supersulfide species are widely distributed in dietary foods, including vegetables, meats, beans, and fermented soybean products (Kasamatsu et al., 2023; Kasamatsu et al., 2024). Although dietary supersulfides are expected to provide health benefits, their direct delivery to target cells remains challenging because of their high reactivity and instability. This limitation highlights the importance of stable dietary precursor molecules that can be metabolized by endogenous enzymes to generate intracellular supersulfides.

Based on these findings, we hypothesized that stable dietary S-alkylcysteines, such as SAC, may serve as outstanding exogenous precursors that undergo CSE-mediated C-S lyase reactions to produce intracellular supersulfides. Therefore, in this study, we aimed to investigate the potential of a series of S-alkylcysteine derivatives as CSE substrates, focusing on the formation of thiol compounds and supersulfides. Through enzymatic and kinetic analyses, we elucidated the metabolic profiles of these compounds and provided novel insights into the molecular mechanisms underlying their physiological activities.

MATERIALS AND METHODS

Materials

The cDNA for rat CSE (pME18S-ratCSE-HA) was a generous gift from Dr. Nozomu Nishi (Life Science Research Center, Kagawa University, Kagawa, Japan) (Nishi et al., 1994). S-methyl-L-cysteine (M1564, LKT Laboratories, St. Paul, MN, USA), S-ethyl-L-cysteine (E1512, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), S-propyl-L-cysteine (BD-A1113642, Ambeed Inc., Arlington Heights, IL, USA), S-allyl-L-cysteine (QA-0046) and S-propargyl-L-cysteine (ST-8604) from Combi-Blocks, Inc. (San Diego, CA, USA), and S-carboxymethyl-L-cysteine (C7757, Sigma-Aldrich, St. Louis, MO, USA) were purchased commercially. Sulfane sulfur probe 4 (SSP4) was obtained from Dojindo Laboratories (SB10, Kumamoto, Japan). All other reagents and materials were obtained from commercial sources and were of the highest quality.

CSE purification

Recombinant rat GST-CSE was expressed in Escherichia coli (DH5α) and purified using glutathione affinity chromatography (Araki et al., 2023). Briefly, cell lysates were applied to a GSH-agarose column (GSH accept, Nacalai Tesque, Kyoto, Japan), and the bound GST-CSE was treated with Turbo3C Protease (Accelagen, Inc., San Diego, CA, USA) to cleave the GST tag. The flow-through containing purified CSE was collected, concentrated, and used in subsequent experiments. Protein levels were measured using the Bradford assay, with bovine serum albumin as the standard (Bradford, 1976).

Measurement of supersulfides

Supersulfides produced by CSE with S-alkylcysteines as substrates were measured using SSP4, a fluorescent probe specific for sulfane sulfurs (Araki et al., 2023). Accordingly, an enzyme concentration of 50 µg/mL in a 20 µL reaction mixture with a 20 min incubation was chosen for the assay. Recombinant CSE (50 µg/mL, 1.1 µM) was incubated in 20 µL of 50 mM HEPES buffer (pH 7.5) containing 50 µM Pyridoxal-5'-phosphate (PLP), either with buffer alone or S-alkylcysteines, for 20 min at 37°C. Samples were diluted five-fold and incubated with 10 µM SSP4 in 100 µL of 20 mM Tris-HCl buffer (pH 7.4) containing 1 mM cetyltrimethylammonium bromide in the dark for 10 min at approximately 25°C. Fluorescence intensity was measured using a microplate reader (Synergy HTX Multi-Mode Microplate Reader, BioTek/Agilent Technologies, Inc., Santa Clara, CA, USA) with excitation at 485/20 nm and emission at 528/20 nm.

Measurement of thiol-containing compounds

Cysteine or thiol-containing compounds produced by CSE from cystathionine or S-alkylcysteines were measured using the 5,5ʹ-dithiobis(2-nitrobenzoic acid) (DTNB; D029, Dojindo Laboratories) assay (Steegborn et al., 1999). The products produced by CSE were measured in 40 mM borate buffer (pH 8.2) containing cystathionine or S-alkylcysteines, 50 µM PLP, and 1 mM DTNB at 30°C for 10 min. The reaction was monitored by recording the increase in absorbance at 412 nm (Synergy HTX Multi-Mode Microplate Reader), which reflected the generation of 2-nitro-5-thiobenzoate anions.

Fluorometric determination of pyruvate

Pyruvate oxidase was used to catalyze the oxidation of pyruvate and the generation of hydrogen peroxide, which subsequently interacted with a fluorescent probe. Measurements were performed using an EnzyChrom Pyruvate Assay Kit (EPYR-100, BioAssay Systems, Hayward, CA, USA). Recombinant CSE (50 µg/mL, 1.1 µM) was incubated in 20 µL of 50 mM HEPES buffer (pH 7.5) containing 50 µM PLP with buffer alone or S-alkylcysteines for 20 min at 37°C. Samples were diluted five-fold and incubated for 10 min at approximately 25°C in 100 µL of the working reagent, following the manufacturer’s instructions. Fluorescence was measured using a microplate reader (Synergy HTX Multi-Mode Microplate Reader) at excitation and emission wavelengths of 530/20 nm and 585/20 nm, respectively.

Statistical analysis

Data are expressed as the mean ± standard error of the mean of at least three independent experiments. Comparisons between two groups were performed using an unpaired Student’s t-test. Statistical analyses were performed using GraphPad Prism (GraphPad Software, Boston, MA, USA).

RESULTS

Structures of S-alkylcysteines and their evaluation as CSE substrates

To investigate whether S-alkylcysteines are substrates for the C-S lyase activity of CSE, we selected a series of cysteine derivatives with varying alkyl chain lengths and degrees of unsaturation, including S-methyl-L-cysteine (SMC; C1), SEC (C2), SPC (C3, saturated), and SAC (C3, unsaturated), which are the major bioactive compounds found in aged black garlic (Fig. 1).

Fig. 1

Chemical structures of S-alkylcysteine derivatives evaluated as CSE substrates. The structures of S-methyl-L-cysteine (SMC), S-ethyl-L-cysteine (SEC), S-propyl-L-cysteine (SPC), S-allyl-L-cysteine (SAC), S-propargyl-L-cysteine (SPRC), and S-carboxymethyl-L-cysteine (SCMC) are shown.

Discrepancy between enzymatic thiol release and supersulfide production

As the direct cleavage product of the C-S lyase reaction is expected to be a free thiol (alkyl mercaptan), we first incubated CSE with each substrate and assessed the thiol compounds generated using DTNB. This assay revealed that SPC produced significant amounts of free thiols (Fig. 2A), indicating CSE-mediated metabolism of this cysteine derivative. Subsequently, we investigated supersulfide production under identical conditions using the SSP4 assay. Supersulfide generation was observed using SEC, SPC, and SAC. In contrast, SMC, S-propargyl-L-cysteine (SPRC), and S-carboxymethyl-L-cysteine (SCMC) exhibited no detectable reactivity in either the DTNB or SSP4 assays (Fig. 2B). Although both free thiols and supersulfides were detected in the reactions with SPC, free thiols were not observed with the SEC and SAC.

Fig. 2

Enzymatic generation of free thiols and supersulfides from S-alkylcysteines by CSE. Recombinant rat CSE was incubated with each substrate (10 mM). (A) Production of free thiol compounds was evaluated using the DTNB assay by measuring the absorbance at 412 nm. (B) Generation of supersulfides was detected using the SSP4 fluorescent probe. ****P < 0.0001 and ***P < 0.001 versus the corresponding control.

Kinetic analysis of CSE using pyruvate and cysteine quantification

To quantitatively evaluate the CSE-mediated α, β-elimination, we measured the production of pyruvate, a direct byproduct of the reaction. The two C3 derivatives that exhibited high substrate reactivity (Fig. 2, SPC and SAC) showed concentration-dependent production of pyruvate (Fig. 3A). The kinetic parameters for each substrate were calculated using Lineweaver–Burk plots (Fig. 3B, C, and Table 1). The Michaelis constant (Km) of SAC was significantly lower than that of SPC (p < 0.05). Furthermore, for cystathionine, enzyme activity was evaluated by measuring cysteine production, which increased in a concentration-dependent manner (Fig. 3D). Kinetic analysis (Fig. 3E) revealed that the Km value for cystathionine was 2.5 ± 0.5 mM, which was similar to that of SAC (3.2 ± 0.6 mM), with no significant difference between the two substrates (P ≥ 0.05).

Fig. 3

Kinetic analysis of CSE-mediated α, β-elimination reactions. (A) Concentration-dependent production of pyruvate from SPC and SAC. Lineweaver–Burk plots for determining the kinetic parameters of (B) SPC and (C) SAC. (D) Concentration-dependent cysteine production from the endogenous substrate cystathionine. (E) Lineweaver–Burk plot of cystathionine.

Table 1. Kinetic parameters of CSE for S-alkylcysteines and cystathionine. The Michaelis constant (Km) and maximum reaction velocity (Vmax) values for SPC, SAC, and cystathionine were calculated based on the Lineweaver–Burk plots shown in Fig. 3. Data are reported as mean ± SEM.

DISCUSSION

In this study, we evaluated the potential of a series of S-alkylcysteine derivatives as CSE substrates. When evaluating the enzymatic generation of free thiols and supersulfides, a significant discrepancy was observed in SEC and SAC. Although free thiols were not observed with SEC and SAC in the DTNB assay (Fig. 2A), supersulfide generation was clearly detected in the SSP4 assay (Fig. 2B). Notably, among the S-alkylcysteine derivatives examined, SAC exhibited the highest supersulfide-producing activity, suggesting that it is a particularly efficient substrate for CSE-mediated supersulfide generation. Although the direct product of the enzymatic reaction is a thiol compound, allyl mercaptan generated from SAC is immediately converted into supersulfides.

Furthermore, kinetic analysis revealed an intriguing relationship between the substrates. Regarding the affinity (Km) for CSE, that of cystathionine is similar to that of SAC, and that of both is greater than that of SPC (Table 1). This result indicates that the presence of a double bond in the allyl group is crucial for enhancing the substrate affinity for CSE and that SAC is recognized and metabolized by CSE with an affinity similar to that of its endogenous counterpart.

Paradoxically, the maximum reaction velocity (Vmax) followed the opposite trend, with cystathionine being faster than SPC, and the Vmax of both was greater than that of SAC (Table 1). This apparent discrepancy may be explained by our previous finding that polysulfides produced during cystine metabolism induce post-translational polysulfidation of CSE, thereby suppressing its enzymatic activity (Araki et al., 2023). Given that SAC produces a larger amount of polysulfides than SPC via the spontaneous disproportionation of its metabolites, the suppression of Vmax for SAC most likely reflects rapid feedback auto-inhibition by the accumulated polysulfides during the assay.

In conclusion, we demonstrated that SAC, a major component of aged black garlic, is an outstanding exogenous substrate (dietary precursor) for CSE, an endogenous supersulfide-producing enzyme. Kinetic analyses revealed that the double bond of the allyl group in SAC is essential for its affinity for CSE, exhibiting a Km value similar to that of cystathionine, the natural physiological substrate. Furthermore, our results suggest that the highly reactive allyl mercaptan generated via the C-S lyase reaction is rapidly converted into supersulfides. A limitation of the present study is that supersulfide production was evaluated using a fluorescent probe-based assay, which cannot unequivocally distinguish or quantify individual supersulfide species. Therefore, future studies will require mass spectrometry-based analyses (e.g., LC-MS/MS) for the direct identification and precise quantification of SAC-derived supersulfide species. Furthermore, elucidation of the molecular structures of these SAC-derived supersulfide species will be essential for understanding the molecular mechanisms underlying the biological activities of SAC. Nevertheless, the present findings indicate, for the first time, that SAC serves as a substrate for CSE, leading to the generation of supersulfides, possibly including allyl hydropersulfide. The present study provides a plausible molecular basis for the reported antioxidant properties of SAC. Taken together, these findings indicate that CSE-mediated intracellular supersulfide production contributes to the multiple physiological and health-promoting benefits of SAC.

ACKNOWLEDGMENTS

We thank Ayaka Yamagishi and Takeru Itoh for their technical assistance in this study. We thank Dr. Tsuyoshi Takata for his valuable comments on the manuscript. We also thank Editage (www.editage.jp) for English language editing.

Funding

This research was funded in part by a Grant-in-Aid for Scientific Research (Early-Career Scientists) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, to S. A. (24K18280). Grant-in-Aid from Showa Pharmaceutical University for Young Scientists [R1-2] (S.A.).

Conflict of interest

The authors declare no competing interests.

Data availability

The datasets generated and/or analyzed in this study are available from the corresponding author upon reasonable request.

Author contributions

Conceptualization: Shoma Araki, Yasuo Watanabe

Data curation: Shoma Araki

Formal analysis: Shoma Araki

Funding acquisition: Shoma Araki

Investigation: Shoma Araki

Methodology: Shoma Araki, Yukihiro Tsuchiya

Project administration: Shoma Araki, Yasuo Watanabe, Yukihiro Tsuchiya

Supervision: Takahisa Nakane, Yasuo Watanabe, Yukihiro Tsuchiya

Validation: Shoma Araki

Visualization: Shoma Araki, Yukihiro Tsuchiya

Writing – original draft: Shoma Araki

Writing – review & editing: Yukihiro Tsuchiya

Ethics approval and consent to participate

Not applicable

Patient consent for publication

Not applicable

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