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. 2023 May 25;145(22):11933–11938. doi: 10.1021/jacs.3c03545

Alkylcysteine Sulfoxide C–S Monooxygenase Uses a Flavin-Dependent Pummerer Rearrangement

Sohan Hazra 1, Tadhg P Begley 1,*
PMCID: PMC10863075  PMID: 37229602

Abstract

graphic file with name ja3c03545_0008.jpg

Flavoenzymes are highly versatile and participate in the catalysis of a wide range of reactions, including key reactions in the metabolism of sulfur-containing compounds. S-Alkyl cysteine is formed primarily by the degradation of S-alkyl glutathione generated during electrophile detoxification. A recently discovered S-alkyl cysteine salvage pathway uses two flavoenzymes (CmoO and CmoJ) to dealkylate this metabolite in soil bacteria. CmoO catalyzes a stereospecific sulfoxidation, and CmoJ catalyzes the cleavage of one of the sulfoxide C–S bonds in a new reaction of unknown mechanism. In this paper, we investigate the mechanism of CmoJ. We provide experimental evidence that eliminates carbanion and radical intermediates and conclude that the reaction proceeds via an unprecedented enzyme-mediated modified Pummerer rearrangement. The elucidation of the mechanism of CmoJ adds a new motif to the flavoenzymology of sulfur-containing natural products and demonstrates a new strategy for the enzyme-catalyzed cleavage of C–S bonds.

Sulfur-containing metabolites are found throughout biological systems, and a diverse array of enzymes has evolved to catalyze the cleavage of carbon–sulfur bonds. Among these enzymes are well-known examples such as PLP-dependent C–S lyases,1,2 redox-neutral S-(2-succino)cysteine lyase,3 radical SAM enzymes,4 SAM-dependent methyltransferases,5 cysteine desulfidases,6 and thioesterases.7 These enzymes use a variety of well-characterized catalytic strategies. Flavoenzymes also play an important role in C–S bond cleavage reactions in the metabolism of diverse compounds. These include S-prenylcysteine,8 alkanesulfonates (SsuD and MsuD),9 dimethyl sulfide (DmoA),10 dimethyl sulfone (SfnG),11 dibenzothiophene sulfone (DszA),12 and N-acetyl-S-(2-succino)cysteine (YxeK).13 Some flavoenzymes, such as S-prenylcysteine lyase, oxidize the C–S bond to a thionium ion, which then undergoes hydrolysis. Others, such as DszA and YxeK, are proposed to use a nucleophilic flavin peroxide to oxidatively cleave the C–S bond.12,13 CmoJ, which is a sequence homologue of YxeK and DszA, is involved in salvaging cysteine from S-alkylated cysteines.14,15

S-Alkylated cysteine is formed in the dealkylation of DNA,16 and in the salvage of cysteine from alkylated glutathione and related low molecular weight thiols involved in the detoxification of environmental alkylating agents.17 The recently discovered cysteine salvage pathway15 shown in Figure 1(14) is widely distributed in soil bacteria. In this pathway, S-alkylated cysteine is acetylated to 2 and oxidized to give sulfoxide 3. CmoJ then catalyzes the cleavage of this sulfoxide to give sulfenic acid 4. Reduction of 4 followed by deacetylation completes the salvage pathway.14 The C–S bond cleavage reaction shown in the conversion of 3 to 4 is an unprecedented flavoenzyme-mediated C–S bond cleavage. This communication describes an experimental strategy to elucidate the mechanism of this reaction.

Figure 1.

Figure 1

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S-Benzyl cysteine salvage pathway in B. subtilis (FRE: Flavin reductase).

In mechanism I, deprotonation of sulfoxide 3 followed by flavin C4a-hydroperoxide (8) mediated hydroxylation forms 9 (Figure 2).14 This then undergoes a facile elimination to give sulfenic acid 4 and benzaldehyde. To test this mechanism, the CmoJ reaction was performed in D2O buffer under anaerobic conditions. In the absence of oxygen, flavin C4a-hydroperoxide (8) would not be formed, thus blocking the hydroxylation reaction. Under these conditions, prolonged incubation of the enzyme and the substrate should provide a sensitive assay for H/D exchange at Cα of the sulfoxide. In the event, LC-MS analysis of the reaction mixture after 2 h of incubation at 37 °C demonstrated that no exchange had occurred (Figure S5). Failure of mechanism I was not entirely unanticipated because the pKa of the benzyl sulfoxide (pKa ∼ 27)18 is well outside the window of accessible deprotonations in biological systems. When this experiment was repeated with the more acidic sulfone 10 (pKa ∼ 20),19 exchange was detected confirming the presence of an active site base close to the Cα of the sulfoxide 3 (Figure S6).

Figure 2.

Figure 2

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Mechanism I for the CmoJ-catalyzed C–S bond cleavage reaction. Inset shows the sulfone analog 10.

Enzymes have evolved three widely used strategies for the catalysis of difficult deprotonation reactions: (1) concerted deprotonation where the proton abstraction is coupled to other chemical steps in the reaction (e.g., deprotonation of a thioester where deprotonation is concerted with carbonyl group protonation);20−22 (2) stabilization of the carbanion intermediate by extensive delocalization (e.g., PLP-dependent enzymes rely on the extensive delocalization of the resulting carbanion to enable deprotonation of α-protons in amino acids);23,24 (3) replacement of the deprotonation by a radical-mediated hydrogen atom abstraction (e.g., radical-mediated epimerization during the biosynthesis of hygromycin and neomycin).25,26 We next turned our focus on evaluating the possibility of a radical-based mechanism for the CmoJ-catalyzed reaction.

A radical mechanism for CmoJ, in which flavin-generated superoxide abstracts a hydrogen atom from the substrate, is shown in Figure 3A. In this mechanism, superoxide 15, generated by the reduction of oxygen by reduced flavin 11, abstracts a hydrogen atom from the substrate to give radical 18. Electron transfer from this radical to the flavin semiquinone followed by the addition of peroxide to the resulting cation gives 20 and regenerates the reduced flavin. Peroxide reduction followed by C–S bond cleavage gives 22 and 4, and elimination of water from 21 regenerates the oxidized flavin. Substrate analog 23 was designed to test for the intermediacy of radical 24 (Figures 3B, S2, S3). With this substrate, the generation of a cyclopropyl carbinyl radical would result in rapid ring opening (rate ∼107s–1)27 to give 26, while nonradical mechanisms would give the cyclopropyl carboxaldehyde 27. In the event, LC-MS analysis of the reaction of 23 with CmoJ followed by derivatization with dansyl hydrazine demonstrated the formation of cyclopropane carboxaldehyde 27 (Figure S9). The ring-opened vinyl sulfoxide 26 was not detected after derivatization with thiophenol (Figure S10).28 In addition, the CmoJ reaction with 23 followed Michaelis–Menten kinetics with no indication of radical-mediated enzyme inactivation (Figures S7, S8). These experiments suggest that the CmoJ-catalyzed C–S bond cleavage reaction does not proceed via a radical intermediate as proposed in Figure 3A.

Figure 3.

Figure 3

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(A) Proposed radical mechanism for the CmoJ-catalyzed reaction with N-acetyl-S-alkyl-l-cysteine sulfoxide 13 (Mechanism II). (B) Proposed cyclopropyl ring-opening (Path 1) and observed products (Path 2).

Previous mechanistic studies on the photooxidation of thioethers suggested a third possible mechanism.29 These studies demonstrated that thioether photooxidation proceeds via a persulfoxide intermediate 28 which then undergoes a Pummerer rearrangement, via intermediate 29, to give α-peroxythioether 30. Intramolecular oxidation of the sulfur via oxathiiranium 31 gives 32 which then fragments to give sulfenic acid 33 and aldehyde 22 (Figure 4A). This chemistry has a striking resemblance to the CmoJ-catalyzed reaction and suggests the mechanistic proposal shown in Figure 4B. Acid-catalyzed elimination of water from sulfoxide 13 generates the thionium ion 34.30 Flavin C4a-hydroperoxide addition to 34 followed by peroxide fragmentation gives oxathiiranium ion 37. Nucleophilic attack by the flavin C4a-hydroxide anion 36 opens the oxathiiranium ring. Elimination of the flavin adduct triggers the cleavage of the C–S bond to form the aldehyde 22 and sulfenic acid 4.

Figure 4.

Figure 4

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(A) Clennan’s mechanism for the C–S bond cleavage in peroxythioethers. (B) Mechanism III for the CmoJ-catalyzed reaction.

Mechanism III is consistent with our previous observation that the aldehyde oxygen is derived from molecular oxygen14 and predicts that the oxygen of the sulfenic acid product 4 is also derived from molecular oxygen and not from the sulfoxide substrate 13. Analysis of the source of the sulfenic acid oxygen is complex because sulfenic acids are highly reactive compounds with both nucleophilic and electrophilic reactivity and undergo rapid dimerization and reduction reactions.31 To facilitate this analysis, the sulfenic acid product was trapped in situ with phenyl vinyl sulfone 40 (PVSu, Figures 5A, and S11, S12).32 Substrate sulfoxide 18O-3, prepared using CmoO and 18O2 (Figure S13), was incubated with CmoJ, and the resulting sulfenic acid was trapped in situ with PVSu. MS analysis demonstrated almost complete (>90%) loss of the 18O label consistent with mechanism III (Figure S14 A).

Figure 5.

Figure 5

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(A) The sulfenic acid trapping strategy, with phenyl vinyl sulfone (PVSu), used in the oxygen labeling experiments. PVSu was found not to inhibit CmoJ. (B) The strategy for the synthesis and trapping of N-acetylcysteine sulfenic acid using the photocaged precursor 42.

To further establish the origin of the sulfenic acid oxygen (water or O2), the CmoJ reaction was run in H218O/H2O (2:1)/16O2. MS analysis showed that 87% of the available 18O was incorporated in the trapped sulfenic acid demonstrating that the sulfenic acid oxygen was derived from the buffer, and not from molecular oxygen (Figures S14B–D, S4). Control experiments demonstrated that the substrate sulfoxide 3 and the trapped product 41 did not exchange substrate 16O with 18O from the buffer (Figure S15).

The literature on the exchange reactivity of sulfenic acids with water is sparse. While sterically protected sulfenic acids do not exchange,33 unhindered sulfenic acids have not been adequately studied. Therefore, the experiments above do not eliminate the possibility that initially formed sulfenic acid exchanges with water before or during the trapping reaction. To evaluate the extent of this exchange we needed access to a clean synthetic sample of N-acetylcysteine sulfenic acid 4.

Our strategy for the synthesis and trapping of N-acetylcysteine sulfenic acid, compatible with the aqueous CmoJ assay conditions, is shown in Figure 5. This strategy involved protecting the sulfenic acid with a photoremovable protecting group (Figure S16).34 Irradiation of 42, in the CmoJ reaction buffer, at 365 nm gave N-acetylcysteine sulfenic acid 4 which was successfully trapped with 10-fold excess PVSu (Figures 5B, S17A–B). When the same reaction was carried out in 18O-buffer, labeled oxygen incorporation into 41 was not observed at high PVSu concentrations (10–20 times substrate, Figure S17C). However, increasing levels of exchange were detected as the concentration of PVSu was decreased (2–5 times substrate, Figure S17D–F, Table S1). This demonstrated that, depending on the exact reaction conditions, N-acetylcysteine sulfenic acid 4 can undergo exchange with water. To determine the origin of the sulfenic acid oxygen in the CmoJ product it was therefore essential to compare the extent of oxygen exchange in sulfenic acid generated under similar conditions in the enzymatic and photochemical systems. This was accomplished by running reactions in which PVSu was in excess (10 mM), substrate concentrations [3] = [42] = 1 mM, and by matching the light intensity and the enzyme concentrations to equalize the rates of N-acetylcysteine sulfenic acid production. Under these conditions, photochemically generated sulfenic acid did not exchange with the buffer containing H218O/H2O (1:1). In contrast, the CmoJ-generated sulfenic acid showed high oxygen atom incorporation from the same buffer (∼80% of available 18O, Figure S18), demonstrating that the oxygen of the CmoJ-generated sulfenic acid was buffer derived as a consequence of the catalytic mechanism and not as an exchange artifact before or during the sulfenic acid trapping. This observation is inconsistent with proposed mechanisms I, II, and III.

Two regio-isomers of the flavin hydroperoxide have been previously characterized differing in the site of attachment of the peroxide to the flavin (9 and 44). A strategy, previously developed to identify the flavin peroxide regio-isomer in the RutA-catalyzed reaction, is shown in Figure 6.35 In this strategy, the enzymatic reaction is run with an inactive substrate analog 2. Under these conditions, the C4a-hydroperoxy flavin eliminates hydrogen peroxide to give oxidized flavin 39. In contrast, the N5-hydroperoxy flavin eliminates water to give the flavin N5-oxide 46 in addition to hydrogen peroxide elimination to give 39. Therefore, the detection of flavin N5-oxide 46 in a flavoenzyme reaction mixture can be used as evidence for the intermediacy of the N5-hydroperoxy flavin. In the event, LC-MS analysis of the reaction mixture resulting from the aerobic incubation of CmoJ with substrate analog 2 and reduced flavin demonstrated the formation of flavin N5-oxide (Figure S19), indicating the likely involvement of N5-hydroperoxy flavin in the CmoJ-catalyzed reaction. However, we cannot yet rule out the possibility that both regio-isomers of the flavin hydroperoxide are catalytically competent.

Figure 6.

Figure 6

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Strategy for the identification of the flavin peroxide regioisomer used in the CmoJ-catalyzed reaction.

In summary, our current mechanism for the alkylcysteine sulfoxide C–S monooxygenase is shown in Figure 7. A concerted elimination of water from 13 followed by the addition of the N5 peroxy-flavin 44 to the resulting thionium ion 34 gives peroxide 48. The regiochemistry of this addition is consistent with a large body of organic chemistry demonstrating exclusive nucleophilic addition to the carbon of thionium ions.30,36−39 Peroxide fragmentation gives oxathiiranium ion 37 and reduced N-5 hydroxyflavin 21. Loss of water from the flavin gives 39, and the addition of water from the buffer to the sulfur of the oxathiiranium ion 37 gives α-hydroxysulfoxide 9 which fragments to form the aldehyde (22) and the sulfenic acid (4) products, respectively. This proposal solves the problem of the unfavorable sulfoxide deprotonation discussed in our previous publication14 and is supported by oxygen labeling experiments which demonstrate that the aldehyde oxygen is derived from molecular oxygen and the sulfenic acid oxygen is derived from the buffer. The absence of CmoJ-catalyzed proton exchange between the substrate and the buffer in the anaerobic reaction (Figure S5) suggests that formation of the flavin hydroperoxide activates the enzyme for the elimination of water from the substrate 13. In addition, the formation of the flavin N5-oxide when the CmoJ reaction is run in the presence of inhibitor 2 is consistent with the assigned regiochemistry of the peroxy flavin 44. To the best of our knowledge, this mechanism describes the first example of an enzymatic Pummerer rearrangement40 as well as a new catalytic strategy for the biochemical cleavage of C–S bonds.

Figure 7.

Figure 7

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Current mechanistic proposal for the CmoJ-catalyzed reaction.

Glutathione or related low molecular weight thiols are widely used for the deactivation of toxic electrophiles, by S-conjugate formation. While it has been reported that such S-conjugates undergo intracellular or extracellular hydrolysis that eventually leads to the formation of cysteine S-conjugates,17,41−43 the fate of these cysteine derivatives was not known. Our previous report14 suggests the role of soil bacteria in the catabolism of such cysteine S-conjugates, by salvaging the cysteine. It had been shown that the pathway utilizes two flavoenzymes (CmoO and CmoJ) to activate and cleave the C–S bond. CmoO oxidizes the thioether to the sulfoxide specifically, while CmoJ performs an oxidative C–S cleavage. In this report, we propose the C–S bond cleavage step proceeds via an oxathiiranium intermediate 37. This discovery adds yet another example to the growing number of new flavin catalytic motifs discovered over the past 20 years.44−53

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c03545.

  • Detailed procedures for the synthesis of all substrates and products, protein purification, enzymatic reaction conditions, and HPLC, NMR, and ESI-MS analysis of enzymatic reactions (PDF)

This research was supported by the Robert A. Welch Foundation (A-0034).

The authors declare no competing financial interest.

Supplementary Material

ja3c03545_si_001.pdf (1.7MB, pdf)

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