Identification and Biosynthesis of 1-Mercaptoethanesulfonic Acid (1

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Identification and biosynthesis of 1-mercaptoethanesulfonic acid (1-MES), an analogue of coenzyme M, found widely in the methanogenic Archaea Robert H White Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00971 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 25, 2017

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Biochemistry

Identification and biosynthesis of 1-mercaptoethanesulfonic acid (1-MES), an analogue of coenzyme M, found widely in the methanogenic Archaea

Robert H. White

Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA

ABSTRACT: Here I report on the identification of 1-mercaptoethanesulfonic acid (1MES), an analog of 2-mercaptoethanesulfonic acid (coenzyme M, HSCoM). 1-MES and HSCoM were both present in the growth media of eight different methanogens at concentrations ranging from ~1-100 µM. In an effort to determine a chemical origin of 1MES several plausible chemical routes were examined each assuming that HSCoM was the precursor. In all examined routes, no 1-MES was formed. However, 1-MES was formed when a solution of vinylsulfonic acid and sulfide were exposed to UV light. Based on these results I conclude 1-MES is formed enzymatically. This was confirmed by growing a culture of Methanococcus maripaludis S2 in the presence of [1,1’,2,2’-2H4]HSCoM and measuring the incorporation of deuterium into 1-MES. 1-MES incorporated three of the four deuteriums from the fed HSCoM. This result is consistent with the abstraction of a C-2 deuterium of the HSCoM, likely by a 5’-dAdoCH2• radical, followed by a radical rearrangement in which the sulfonic acid moves to the C-1 position, followed by abstraction of a H• likely from 5’-dAdoCH2D. At present the reason for the

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production of 1-MES is not clear. This is the first report of the occurrence of 1-MES in Nature.

Introduction Coenzyme M (2-mercaptoethanesulfonic acid, HSCoM) was one of the first coenzymes to be identified to function in methanogenesis.1 HSCoM is found in methanogenic Archaea, a group of microorganisms that are major players in the global carbon cycle, producing ~109 tons of methane annually.2, 3 The terminal step in this process involves the reduction of the S-methyl derivative of HSCoM which serves as the substrate supplying the methyl group to the Ni of F430 coenzyme as part of the mechanism of the methyl coenzyme M reductase reaction leading to methane formation.4, 5 Only a few other molecules, such as 3-(methylthio)propionic acid, can serve as an alternate substrate for this enzyme.6 Known examples of the excretion of small molecules, other than methane, by methanogens is sparse with only the excretion of methionine by M. volta being described.7 One explanation for this is that these cells energetically live on the edge of survival and thus just do not produce excess metabolites. Yet they can secrete natural products when genes for their biosynthesis are added to their genomes.8 They do not grow using typical bacterial fermentation pathways thus the products of such processes are not secreted. They also do not produce antibiotics or secondary natural products on the bases of their genomic sequences. During our work to establish the origin of the sulfite required for the biosynthesis of HSCoM,9 we developed a method for measuring the incorporation of 34S-sulfate into

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Biochemistry

HSCoM using M. maripaludis S2 cells growing on McN media containing 34S-sulfate. HSCoM was detected in both the cell extracts and in the media after the growth of the cells. These analyses were accomplished by derivatization of the thiols with monobromobimane (mBBr), purification of the resulting derivative by preparative TLC, followed by ultra performance-liquid chromatography-high resolution-electrospray-mass spectrometry (UPLC-HR-ESI-MS) to identify the thiol containing products. The procedure used was basically those described for the recent identification of 3mercaptopropionic acid (MPA) in growth media of methanogens.10 The results of these experiments failed to show the presence of any labeled HSCoM in the cells but did demonstrate the presence of HSCoM in the growth media. To my surprise, however, I identified two peaks with the exact same mass as the HSCoM derivative (333.0540). The new peak eluted after a known sample of HSCoM. The only possible stable chemical structure for this observed molecule was 1-mercaptoethanesulfonic acid (1MES). 1-MES was confirmed by its chemical synthesis and mass spectral analysis. Here I report on the identification of this new compound and its presence in the growth media of eight different methanogens. I present data showing that 1-MES is derived from [1,1’,2,2’-2H4]-HSCoM with the loss of one deuterium, suggesting it is produced enzymatically by a SAM radical enzyme. Why 1-MES is made by these methanogens remains to be established. MATERIALS AND METHODS Chemicals. 1-Chloroethane-1-sulfonyl chloride was obtained from Enamine. Bromobimane (mBBr), and vinylsulfonic acid sodium salt were obtained from SigmaAldrich. [1,1’,2,2’-2H4]-Dibromoethane was obtained from MSD isotopes.

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Synthesis of 2H4-HSCoM. [1,1’, 2,2’]-Dibromoethane ( 98% 2H4) was reacted with sulfite to generate [2H4]-2-bromoethanesulfonic acid as previously described11 and product was crystalized from ethanol. The deuterated sulfonic acid (21 mg, 0.1 mmol) was then mixed with 0.2 ml of 1 M NaSH under nitrogen at RT and the reaction followed by TLC. After three days at 50 °C, TLC analysis using solvent system [acetonitrile−water−formic acid (88%) (19:2:1 vol/vol/vol)] showed that all the reactant had been converted into HSCoM. The concentration of HSCoM in the sample was analyzed by isotope dilution using a known sample of unlabeled HSCoM. A 5 µl portion of the labeled sample was mixed with 5 µl of a 1 M solution of unlabeled HSCoM in water, acidified with 5 µl of 1 M HCl, and then the sample was evaporated to remove the excess sulfide. The HSCoM was then reacted with mBBr to form the bimane (B) derivative and from the measured ratio of labeled to unlabeled sample determined by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), the concentration of the labeled sample was determined to be 0.37 M. The entire sample was then acidified, evaporated to remove excess sulfide and then dissolved in 0.2 mL of water. This sample was then used for the feeding experiments (described below). Synthesis and analysis of 1-MES. 1-MES was prepared from 1-chloroethane-1sulfonyl chloride following previously described methods.12 The compound was converted to the B-derivative and purified by preparative TLC as previously described.10 Analysis of methanogenic cell extracts for thiols. Samples were assayed for the presence of thiol containing compounds using both HPLC with fluorescent detection and by LC-ESI-MS as preciously described.10 The methods were developed following those described by Fahey,13 which have also been modified for the analysis of sulfite

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Biochemistry

and thiosulfate in sea water14 and for the identification of 3-mercaptopropionic acid (MPA) in methanogens.10 Cell lysates were obtained via sonication of cell pellets in buffer as previously described and contained ~20 mg/ml of protein.15 A 100 µl sample of this cell lysate was mixed with 120 µl of methanol and centrifuged (14000 × g; 10 min) to remove the large biomolecules. The supernatant was separated and the volume was reduced to ~20 µl by evaporation with a stream of nitrogen gas. To the concentrated sample, 20 µl of 50 mM tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) (adjusted to pH 7 with NaOH), 20 µl of 0.1 M borate buffer (pH 9.3), and 20 µl of 0.1 M solution monobromobimane (mBBr) in acetonitrile was added. After 15 min at room temperature, the mBBr derivatives (B-) were purified by preparative TLC using solvent system [acetonitrile−water−formic acid (88%) (19:2:1 vol/vol/vol)] where BHSCoM had a Rf = 0.43 and B-1-MES had a Rf = 0.45. The samples were then eluted from the TLC plate with 50% methanol in water and analyzed by HPLC-UV-ESI-MS/MS. Analysis of growth media for the presence of HSCoM and 1-MES. Growth media from the growth of the cells shown in Table 1 were supplied by Dr. Seigo Shima in Malburg, Germany. Cells were grown in the media referenced in Table 1. Two milliliters of each media sample free of cells was placed in a vial and 4 mg of TCEP HCl was added. The solution was brought to pH ~3-4 by the addition of 1 M HCl and then concentrated at ~90 °C with stream of nitrogen gas to ~ 0.5 mL at which point the salts present in the media appeared. This procedure allowed for the removal of sulfite and sulfide by evaporation and also reduced any disulfide that may have formed due to air oxidation. To the resulting solution was added 50 µl of 0.1 M sodium borate buffer (pH 9.3) and the sample titrated to pH ~9.3 with the addition of 1 M NaOH. Generally, these

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procedures produced a slightly cloudy solution to which 100 µl of a 0.037 M solution of mBBr in acetonitrile was added. After 1 hr at RT the sample was acidified to pH ~1 by the addition of 6 M HCl (~20 µl) and the whole sample was applied to a C18 column (4 x 20 mm) which was washed with 1 mL of 10 mM HCl and the sample eluted with 50% methanol in water. (The addition of the B to these molecules allowed them to be retained on the C18 column.) This fraction was evaporated to dryness and the B-HSCoM and B-1-MES were purified by preparative TLC as described above. Both areas of the plates were removed together in one band for analysis. Control assays were done on the media before it was inoculated with cells. HPLC analysis with fluorescent detection. A Shimadzu HPLC system with a Pursuit XRs 5 C18 (Agilent, 250 x 4.6 mm, 2,6 um particle size) equipped with a photodiode array detector (PDA) and a fluorescence detector was utilized for initial identification of various bimane derivatives. The elution profile consisted of 5 min at 95% buffer A (25 mM sodium acetate pH 6.0, 0.02% sodium azide) and 5% methanol, followed by a linear gradient from 5% to 50% methanol over 25 min at 1 mL/min. The Bderivatives were detected with excitation = 400 nm and emission = 490 nm. B-HSCoM eluted at 16.992 min and B-1-MES eluted at 17.268 min. HPLC-UV-ESI-MS/MS analysis. An AB Sciex 3200 Q Trap mass spectrometer attached to an Agilent 1200 series liquid chromatograph with a Kromasil 100-5-C18 column (4.6 x 250 mm) and a UV diode array detector was used for the identification of the peaks, and for the mass analysis of thiol compounds. Solvent A was 25 mM ammonium acetate and solvent B was 100% methanol. The flow rate was 0.7 mL/min and the elution profile consisted of a 3-min wash at 100% A, followed by a 15 min linear

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Biochemistry

gradient to 65% B. The injection volume used was 15 µL. Electrospray ionization was employed at 4500 volts and a temperature of 600 °C. Curtain gas, gas 1, and gas 2 flow pressures were 35, 60, and 50 psi, respectively. Desolvation, entrance, and collision cell potentials were 35, 10, and 30 volts, respectively. Product ion scans of various Bderivatives indicated that a common product ion, 192 m/z, was present in all spectra analyzed. Therefore, a precursor ion scan method was developed to identify molecules that fragmented to yield this ion. This technique first scans Q1, in this case over a mass range of m/z 150-650 in 0.75 seconds, and then ions are subjected to collisionally induced dissociation (CID) in Q2. Q3 is set to pass only the selected fragment mass of interest, 192 m/z for this experiment. When the 192 mass is detected, the mass going into the Q2 is recorded to reveal the (M + H)+ mass. Analyst software (Applied Biosystems/MDS SCIEX) was used for system operation and data processing. Ultra performance-liquid chromatography-high resolution-electrospraymass spectral (UPLC-HR-ESI-MS) analysis of samples. TLC purified fractions containing the B-HSCoM and B-1-MES were analyzed on a Waters SYNAPT G2-S high definition mass spectrometer connected to a Waters Acquity UPLC I-class system with an Acquity UPLC BEH C18 (Waters, 2.1 mm x 75 mm, 1.7 um particle size) column. Solvent A was 0.1% formic acid in water and solvent B was 100% acetonitrile. The flow rate was 0.2 mL/min and gradient elution was employed in the following manner (t (min), %B): (0.01, 5), (6, 15), (21, 35), (23, 65). Ten µl of sample was injected for each sample analyzed. The mass spectral data were collected in high resolution MSe continuum mode. A lock spray scan (Function 3) was collected every 20 seconds for calibration and the lock spray analyte used was Leucine-Enkephalin. Parameters were:

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2.8 kV capillary voltage, 125 °C source temperature, 350 °C desolvation temperature, 35 V sampling cone, 50 l/h cone gas flow, 500 l/h desolvation gas flow, and 6 l/h nebulizer gas flow. The collision energies for the low energy scans (Function 1) were 4V and 2V in the trap region and the transfer region, respectively. Collision energies for the high energy scans (Function 2) were ramped from 25 to 45 V in the trap region and 2V in the transfer region. Data were analyzed using MassLynx program (Waters). Testing non-radical routes for the formation of 1-MES. I tested three different chemical routes for the possible formation of 1-MES. These tests served not only as controls to test for 1-MES formation abiotically, but also to give us insights into possible chemical or enzymatic routes for its formation. First, the addition of sulfide to vinylsulfonic acid was examined to determine if it was possible for sulfide to add to both the 1 and 2 positions of vinylsulfonic acid to produce HSCoM and 1-MES, respectively. A 20 µl aliquot of a 0.52 M (25 wt %) solution of vinylsulfonic acid sodium salt was mixed with 60 µl of 1 M sodium sulfide at pH 7.0 under argon and the sample heated at 80 °C for 12 hr. A 10 µl aliquot of the sample was removed and mixed with 25 µl of 1 M HCl and the sample evaporated to remove any hydrogen sulfide. The resulting sample was dissolved in borate buffer and analyzed as described above for the presence of HSCoM and 1-MES. Second, I examined if 1-MES could be formed from HSCoM added to growth media containing sulfide. Thus, HSCoM at concentration of a 5 mM was added to fresh sterile anaerobic McN growth media and incubated for 4 days at RT and then the sample, which showed no cell growth, was assayed for the presence of 1-MES. Finally, a 100 µl of 5 mM solution of HSCoM was heated in water at 100 °C for 12 hr with 5 mg of S8 and assayed for 1-MES formation to determine if 1-MES was formed.

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Biochemistry

Photochemical formation of HSCoM and 1-MES from vinylsulfonic acid. On the basis of the well-known photo-addition of hydrogen sulfide to olefinic bonds,16 I tested this as a possible route to 1-MES. Thus to 1 mL of a 0.1 M anaerobic aqueous solution of sodium vinylsulfonate was added 100 µl of a pH 7.0 anaerobic 1 M solution of sodium sulfide and the sample placed in a 1 mL 1 cm UV-visible spectrometer cell capped with a septum under nitrogen gas. The colorless sample was then exposed to a short wavelength UV light from a model UVGL-2S mineralight lamp for 12 hr at RT. The resulting clear warm yellow solution was removed and acidified by the addition of 100 µl of 6 M HCl. This resulted in the generation of a white precipitate of elemental sulfur and the loss of the yellow color. The formation of the sulfur resulted from the disproportionation of some of the sulfide to sulfur and hydrogen.17 The sulfur was removed by centrifugation (10,000 x g, 5 min) and the resulting separated colorless sample was evaporated 2x to dryness with a stream of nitrogen gas to remove sulfide. The resulting colorless solid was dissolved in 200 µl of water and 50 µl of 0.1 borate buffer pH 9.2 and 4 mg of TCEP HCl was added and the sample adjusted to pH 9.2 and the B-derivatives prepared and purified as described above. Testing the incorporation of labeled HSCoM into 1-MES. To test if HSCoM was a precursor to 1-MES we grew a 5 mL culture of M. maripaludis S2 in the presence of [1,1,2,2-2H4]-HSCoM (5 mM) in McN media18 for 12 hr at 37 °C. After growth, the cells were separated by centrifugation (31000 x g,10 min) and 2 mL of the media was assayed for the presence of both labeled and unlabeled HSCoM and 1-MES as described above. RESULTS AND DISCUSSION

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Discovery of 1-MES, an isomer of HSCoM. Analysis of the media for HSCoM after the growth M. maripaludis S2 revealed two chromatographic peaks for the purified B-HSCoM fraction. The first one coeluted by LC-MS and HPLC with fluorescence detection with a known sample of B-HSCoM. The second peak had exactly the same mass and isotopic peak cluster as B-HSCoM. On the Synapt LC-HR-ESI-MS system in the low energy scans, the first peak eluted at 6.08 min and the second peak eluted at 6.41 min (Figure 1). The calculated mass for the (MH)+ for the B-derivatives of HSCoM and 1-MES is 333.0579 and the measured masses were both 333.0573. Further support for the assigned structures (Figure 2) were obtained from the 34S isotope peaks. The calculated mass of the 34S containing isotope peak was 335.0511 and the measured was 335.0510. The intensity of this peak was 9.5% of the MH+ ion showing that both molecules contained two sulfur atoms. Mass scans were done to correct for any background ion intensities for each ion. Each isomer also showed MNa+ and MK+ ions at 355.0355 and 371.0043, respectively. The intensity of the 34S isotope peak of these metalated was 9.5% of each ion, again demonstrating that both molecules contained two sulfur atoms. The percent intensities of these metalated ions to the respective MH+ were 50% for the MNa+ and 30% for the MK+ ions for HSCoM and 28% for the MNa+ and 16% for the MK+ ions for 1-MES. These results further confirm that the observed compounds were not identical molecules.

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Biochemistry

Figure. 1. UPLC-HR-ESI-MS analysis of the sample containing B-HSCoM (first peak) and B-1-MES (second peak) from M. maripaludis media showing the 333.0573 ion trace.

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Figure 2. Chemical structures of mBBr derivatives of B-HSCoM and B-1-MES and possible sources of unique fragment ions in their ESI-MS/MS spectra.

Further evidence that the two compounds were not identical came from the high energy scans to obtain MS/MS fragmentation of the MH+ ions at 333.0573. Most of the observed fragments from each molecule were identical with the exact same masses indicating that the molecules were very similar. The major differences observed, were a

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Biochemistry

much more intense 225.0724 fragment ion from B-1-MES than from in B-HSCoM, and the presence of a 224.0654 ion in B-HSCoM that was not present in B-1-MES. B-1-MES also showed a MH+− 26.9960 (CO) at 305.0656 that was not observed in B-1-MES. Elimination of possible abiotic chemical routes for the formation of 1-MES. In order to determine how 1-MES might be formed, several attempts were taken to determine a possible abiotic route considering that the precursor was HSCoM. One possibility examined was that HSCoM eliminates hydrogen sulfide to form vinylsulfonic acid, which then reacts with sulfide to form 1-MES as shown in Figure 3. Heating vinylsulfonic acid with sulfide led only to the formation of HSCoM which would be the expected isomer for a Michael adduct. This was not consistent with the route proposed in Figure 3. Next, I tested if 1-MES could be formed from HSCoM added to growth media containing sulfide. HSCoM at 5 mM was added to fresh growth media used to grow M. maripaludis and it was incubated for 4 days at RT and then the sample was assayed for the generation of 1-MES. Only HSCoM was detected. Finally, I tested if persulfides could be involved in the conversion. HSCoM was heated in water with a suspension of S8 at 100 °C for 4 hr. Again, no formation of 1-MES was observed indicating that persulfides were not likely involved in the conversion.

H2S

H2S

HS-CH2CH2-SO3

CH2=CH-SO3

coenzyme M

vinylsulfonate

SH CH3CH-SO3 1-mercaptoethanesulfonic acid (1-MES)

Figure 3. A simple chemical route to 1-MES involving the elimination of sulfide followed the readdition sulfide to the 1 position of vinylsulfonate.

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Testing radical reactions for the formation of 1-MES from HSCoM. The reaction of alkenes with sulfide can proceed either by radical16, 19 or electrophilic additions.20 I investigated if the reaction of vinylsulfonic acid with sulfide in the presence of UV would lead to the production of 1-MES. This experiment resulted in the production of about equal amounts of HSCoM and 1-MES when assayed by LC-MS of the mBBr derivatives. This result indicated that radical reactions could produce 1-MES. Exposure of HSCoM to sulfide in the presence UV did not produce 1-MES indicating that under these conditions no photochemical route existed for the conversion of HSCoM to 1MES. On the basis of these data I concluded that vinylsulfonic acid was not a likely intermediate in the conversion of HSCoM to 1-MES. To establish if HSCoM was a precursor to 1-MES, M. maripaludis S2 was grown in the presence of [1,2-2H4]coenzyme M (5 mM) and the incorporation of label into 1-MES was measured. The observed incorporation of the label HSCoM into 1-MES resulted in the production of 1MES that contained 3.9% of the molecules with 2H3 and less than 0.4% of 1-MES with 2

H4. The amount of unlabeled 1-MES detected was 4% of the amount of [2H4]-HSCoM

detected. A small number of molecules with 2H1 and 2H2 (< 0.4%) were observed but could be explained by their occurrence in the fed HSCoM. As a result of the large amount of labeled HSCoM added to the media only 0.3% of the HSCoM recovered from the media was not labeled and was from the HSCoM produced by the cells. I propose that these results are best explained by an enzyme-catalyzed radical rearrangement.

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Biochemistry

Currently the closest example of such a radical reaction can be found in lysine 2,3-aminomutase (LAM)21 where a hydrogen from C-3 of a PLP bound α-lysine is abstracted by 5’-dAdoCH2• to generate the C-3 radical on the α-lysine and 5’-dAdoCH3. The α-lysine then undergoes a radical rearrangement to produce a PLP bound β-lysine containing a radical at C-2 which then abstracts H• from 5’-dAdoCH3 to regenerate a PLP bound β-lysine and 5’-dAdoCH2•. The β-lysine is then released from the PLP and the reaction can start again after the binding of α-lysine. A analogous P450 radical rearrangement has been described.22 On the basis of these data I propose that the enzymatic reaction occurs as shown in Figure 4 and is catalyzed by a SAM radical enzyme. Here the required radical rearrangement would begin by the abstraction of the C-2 hydrogen by 5’-dAdoCH2• to generate the C-2 radical on the HSCoM that then undergoes a rearrangement where the sulfonic acid moves to the original C-1 position with the radical appearing at original C-2. This substrate radical then reacts with 5’-dAdoCH3 to regenerate 5’-dAdoCH2•. Such a regeneration of would allow for the non-stoichiometric or catalytic use of SAM.

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O

S

HS

H

HS O

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C D D

D

2 D

5’-dAdoCH2

O 4)

1-mercaptoethanesulfonic acid (1-MES)

O

S

2-mercaptoethanesulfonic acid (HSCoM)

5’-dAdoCH2D HS

HS O

1)

D D

D

D

D

D D C D 1 S O O O

D S

O

O O

O 2)

3) HS

D D

D S O

O

Figure 4. A possible radical rearrangement responsible for the conversion of HSCoM to 1-MES.

In order to explain the incorporation of only three deuteriums into 1-MES from HSCoM by the growing cells of M. maripaludis S2 one must consider possible kinetic isotope effects, randomization of the deuterium with the two hydrogens on the methyl group of 5’-dAdoCDH2, as well as substrate exchange on the enzyme. The kinetic isotope effects could occur at two different steps. The first step would involve the removal of the deuterium from the C-2 carbon of the substrate (step 1, Figure 4). Having a large kinetic isotope effect would discriminate against the use of the labeled substrate. If the amount of the labeled HSCoM in the cells is low this would allow for the preferred use the unlabeled HSCoM to produce unlabeled 1-MES. In the case of propane-1,2diol dehydratase the deuterium isotopic effects on the removal of the 1-2H was measured at ~12.23 The effect here would be a lowering of the incorporated of any label

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into 1-MES. The second kinetic isotope effect would come into play during the removal of the hydrogen/deuterium from AdoCH2D (Figure 4, step 4). If no isotope effect was present then one would have only a 30% chance that a deuterium would be reintroduced back into 1-MES. If the deuterium kinetic isotope effect was 5, then the transfer of a H would be 10 times more likely than a deuterium. This could account for our observed the loss of one of the deuteriums. An alternate explanation is that the H• that is removed by 5’-dAdoCH2• is exchanged with non-labeled protein hydrogens before it is reincorporated back into the product, as shown in Figure 5. Such an exchange is observed to occur in the SAM radical spore photoproduct lyase (SPL).24 This SAM radical enzyme abstracts the 6proR hydrogen from spore photoproduct with 5’-dAdoCH2•.25 The radical product then rearranges to cleave a C-C bond and the resulting radical, now residing on a tyrosine, is Combined with hydrogen radical derived from a conserved cysteine26 to form the TpT product with a hydrogen, and a cysteinyl radical on a conserved cysteine. Radical II in Figure 5 would be expected to be very reactive since it has no way to delocalize the unpaired electron 27 and thus this reactive radical could abstract a H• from a thiol to produce the 1-MES product. A third possibility is that the generated 5’-dAdoCH2• is used stoichiometrically and the product radical is converted to 1-MES by a mechanism not involving the regeneration of 5’-dAdoCH2•.

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Page 18 of 25

SAM HS

[4Fe-4S]2+

D

D

[4Fe-4S]+

C D

D

HS

S O O

O

Met + Y-OH

5’-dAdoCH2

2-mercaptoethanesulfonic acid (HSCoM)

5’-dAdoCH2D

D

D

HS

O S O O

D


HS

D O

3

S O

D

C-SH

D

2

D

4

Y-O

D

D S O

C-S

?

1

HS

O

D exchanges with water

O

H

O

O

D D

S

D

O radical II

radical I

Figure 5. Possible explanation for the loss of one deuterium from [1,1’,2,2’-2H4]HSCoM during its conversion to 1-MES based on a photolyase model. Identification and concentrations of HSCoM and 1-MES in different methanogens. The concentrations for HSCoM in the media after cell growth ranged from 1.5 to 18 µM and the 1-MES concentrations ranged from 110 to 0.086 µM for the methanogens listed in Table 1. An unknown portion of these concentrations could be due to cell lysis which is known to occur in the methanogens.28 Neither HSCoM or 1MES was detected in the media from Methanosarcina acetivorans before or after growth. This observation may be connected to the fact that the pathway for HSCoM biosynthesis in the Methanosarcinales, of which M. acetivorans is a member, has an alternate pathway for HSCoM biosynthesis.29 The discovery 1-MES was unexpected since this compound has never been detected before as a natural product. In addition, it has also never been observed that the methanogens secrete HSCoM into the media. The average concentrations of HSCoM measured in the media was ~ 11 µM. A

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Biochemistry

transport system is known for HSCoM and may be used to transport these compounds out of the cells into the media.30

Table I. Analysis of the concentrations HSCoM and 1-MES excreted into their grown by various methanogens

Methanogen grown in salts media with added sulfide and/or cysteine

Concentration in the media after growth (µM) HSCoM 1-MES

Media18

Not detected

Methanococcus maripaludis18

18

6.8

Methanothermobacter thermautotrophicus ∆H31

7.1

19

Methanothermobacter wolfeii32

44

1 12

Methanothermococcus thermolithotrophicus33

8.1

5.8

Methanoculleus CR-1 (DSMZ media 141)

0.53

0.47

Methanospirillum hungatei JF134 4.0 Methanospirillum hungatei GP134 6.8 Methanocorpusculum labreanum 1.5 (DSMZ media 119)

7.3 0.086 0.65

Identification of an enzyme likely responsible for 1-MES formation. In order to find the enzyme responsible for the formation of 1-MES we searched for a SAM radical enzyme that is genomically connected to the genes involved in the biosynthesis of HSCoM. These included CoMA, CoMB, CoMC, and CoMDE9 as well as CoMF (MMP1603) a homolog of MJ0099, the enzyme responsible for catalyzing the

19


Biochemistry

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generation of homocysteine from aspartate semialdehyde and sulfide.35 This reaction is analogous to the proposed final reaction in HSCoM biosynthesis catalyzed by CoMF.9 I am adding CoMG (MMP0270) to this list for the enzyme catalyzing 1-MES formation from HSCoM. Each of these genes were connected by co-occurrence in all the methanogenic genomes and in the genomes of the Methanomicrobiales, Methanosphaerula palustris, Methanoplanus petrolearius, Methanoculleus marisnigri, Methanocorpusculum labreanum and Methanospirillum hungatei the CoMG, HSCoMF and CoME were each in this order in an operon. As no homolog of MMP0270 was seen in Methanosarcina acetivorans this is consistent with the absence of 1-MES in this organism. CoMG, however, does not have the signature SAM radical motif but does have a conserved CX2CX4C motif as recently identified in the radical SAM enzyme thiamin pyrimidine synthase.36

Function of 1-MES. I propose that 1-MES along with the different modified F430 coenzymes identified in the methanogens37 are used to allow for the anaerobic oxidation of methane (AOM) by the methanogens as first established in 2005.38 Recent work has confirmed that a genetically modified Methanosarcina acetivorans can in fact grow by the AOM.39 The involvement of F430-3 in the AOM by ANME-1 and/or ANME-2 has been indicated by the presence of this modified F430 coenzyme in the methylcoenzyme M reductase (MCR) isolated from these cells present in microbial mats from the Black Sea.40 I speculate that these modified forms of F430 and 1-MES are used to alter the directionality of the MCR reaction. It is also possible that HSCoM and/or 1-MES secreted into the media, are used to bind toxic heavy metals to prevent them from interfering with cell growth. HSCoM has

20


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Biochemistry

been shown to form Complexes with Rh and Tc41 and bismuth.42 These metal as well as other heavy metals are present in the vents where these cells grow. Vent fluids and plume typically have 1-3 orders of magnitude higher heavy metal concentrations than sea water.43 As expected sulfide also ameliorates metal toxicity for deep-sea hydrothermal vent archaea 44 by precipitation of the metal sulfides. AUTHOR INFORMATION Corresponding author E-mail: [email protected]. Phone: 540-231-6605. Fax: 540-231-9070. ORCID Robert H. White: 0000-0002-9105-7445 Funding This work was supported by the National Science Foundation Grant MCB1120346.

ACKNOWLEGMENTS The authors thank Dr. W. Keith Ray for performing the mass spectrometry experiments using Synapt mass spectrometer. The mass spectrometry recourses are maintained by the Virginia Tech Mass Spectrometry Incubator, a facility operated in part through funding by the Fralin Life Science Institute at Virginia Tech and by the Agricultural Experiment Station Hatch Program (CRIS Project Number: VA-135981). I would like to think Dr. William Whitman for growing the culture of M. maripaludis S2 in the presence of [1,2-2H4]-HSCoM. I would also thank Danielle Miller and Water Niehaus for help in editing the manuscript. ABBREVIATIONS

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UPLC-HR-ESI-MS,

Ultra

performance-liquid

Page 22 of 25

chromatography-high

resolution-

electrospray-mass spectral; HSCoM, coenzyme M; 1-MES, 1-mercaptoethanesulfonic acid, TCEP HCl; tris-(2-carboxyethyl)phosphine hydrochloride ; monobromobimane, B. REFERENCES

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[14] Rethmeier, J., Rabenstein, A., Langer, M., and Fischer, U. (1997) Detection of traces of oxidized and reduced sulfur compounds in small samples by combination of different high-performance liquid chromatography methods, J Chromatogr A 760, 295-302. [15] White, R. H., and Xu, H. (2006) Methylglyoxal is an intermediate in the biosynthesis of 6deoxy-5-ketofructose-1-phosphate: a precursor for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii, Biochemistry 45, 12366-12379. [16] Vaughan, W. E., and Rust, F. F. (1942) The photo-addition of hydrogen sulfide to olefinic bonds, Journal of Organic Chemistry 7, 472-476. [17] Hara, K., Sayama, K., and Arakawa, H. (1999) UV photoinduced reduction of water to hydrogen in Na2S, Na2SO3, and Na2S2O4 aqueous solutions, J Photoch Photobio A 128, 27-31. [18] Sarmiento, F., Leigh, J. A., and Whitman, W. B. (2011) Genetic systems for hydrogenotrophic methanogens, Methods Enzymol. 494, 43-73. [19] Griesbaum, K. (1970) Problems and Possibilities of Free-Radical Addition of Thiols to Unsaturated Compounds, Angewandte Chemie-International Edition 9, 273-+. [20] Prilezhaeva, E. N., and Shostakovskii, M. F. (1963) The Thiylation of Olefins, Russ Chem Rev 32, 399-426. [21] Horitani, M., Byer, A. S., Shisler, K. A., Chandra, T., Broderick, J. B., and Hoffman, B. M. (2015) Why Nature Uses Radical SAM Enzymes so Widely: Electron Nuclear Double Resonance Studies of Lysine 2,3-Aminomutase Show the 5'-dAdo* "Free Radical" Is Never Free, J Am Chem Soc 137, 7111-7121. [22] Ortiz de Montellano, P. R., and Nelson, S. D. (2011) Rearrangement reactions catalyzed by cytochrome P450s, Arch Biochem Biophys 507, 95-110. [23] Frey, P. A. (2014) Travels with carbon-centered radicals. 5'-deoxyadenosine and 5'deoxyadenosine-5'-yl in radical enzymology, Acc. Chem. Res. 47, 540-549. [24] Yang, L., and Li, L. (2015) Spore photoproduct lyase: the known, the controversial, and the unknown, J Biol Chem 290, 4003-4009. [25] Yang, L., Lin, G., Liu, D., Dria, K. J., Telser, J., and Li, L. (2011) Probing the reaction mechanism of spore photoproduct lyase (SPL) via diastereoselectively labeled dinucleotide SP TpT substrates, J Am Chem Soc 133, 10434-10447. [26] Benjdia, A., Heil, K., Winkler, A., Carell, T., and Schlichting, I. (2014) Rescuing DNA repair activity by rewiring the H-atom transfer pathway in the radical SAM enzyme, spore photoproduct lyase, Chem Commun (Camb) 50, 14201-14204. [27] Viehe, H. G., Janousek, Z., Merenyi, R., and Stella, L. (1985) The Captodative Effect, Acc. Chemi. Res. 18, 148-154. [28] Newbold, C. J., Ushida, K., Morvan, B., Fonty, G., and Jouany, J. P. (1996) The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid, Lett Appl Microbiol 23, 421-425. [29] Graham, D. E., Taylor, S. M., Wolf, R. Z., and Namboori, S. C. (2009) Convergent evolution of coenzyme M biosynthesis in the Methanosarcinales: cysteate synthase evolved from an ancestral threonine synthase, Biochem. J. 424, 467-478. [30] Dybas, M., and Konisky, J. (1989) Transport of coenzyme M (2-mercaptoethanesulfonic acid) and methylcoenzyme M [(2-methylthio)ethanesulfonic acid] in Methanococcus

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voltae: identification of specific and general uptake systems, J. Bacteriol. 171, 58665871. [31] Balch, W. E., Fox, G. E., Magrum, L. J., Woese, C. R., and Wolfe, R. S. (1979) Methanogens: reevaluation of a unique biological group, Microbiological reviews 43, 260-296. [32] Schmitz, R. A., Albracht, S. P., and Thauer, R. K. (1992) A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei, Eur J Biochem 209, 1013-1018. [33] Belay, N., Sparling, R., and Daniels, L. (1986) Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus, Appl Environ Microbiol 52, 1080-1085. [34] Ferry, J. G., Smith, P. H., and Wolfe, R. S. (1974) Methanospirillum, a New Genus of Methanogenic Bacteria, and Characterization of Methanospirillum-Hungatii Sp-Nov, International journal of systematic bacteriology 24, 465-469. [35] Allen, K. D., Miller, D. V., Rauch, B. J., Perona, J. J., and White, R. H. (2015) Homocysteine is biosynthesized from aspartate semialdehyde and hydrogen sulfide in methanogenic archaea, Biochemistry 54, 3129-3132. [36] Fenwick, M. K., Mehta, A. P., Zhang, Y., Abdelwahed, S. H., Begley, T. P., and Ealick, S. E. (2015) Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase, Nat Commun 6, 6480. [37] Allen, K. D., Wegener, G., and White, R. H. (2014) Discovery of multiple modified F430 coenzymes in methanogens and anaerobic methanotrophic archaea suggests possible new roles for F430 in nature, Appl. Environ. Microbiol. 80, 6403-6412. [38] Moran, J. J., House, C. H., Freeman, K. H., and Ferry, J. G. (2005) Trace methane oxidation studied in several Euryarchaeota under diverse conditions, Archaea 1, 303-309. [39] Soo, V. W., McAnulty, M. J., Tripathi, A., Zhu, F., Zhang, L., Hatzakis, E., Smith, P. B., Agrawal, S., Nazem-Bokaee, H., Gopalakrishnan, S., Salis, H. M., Ferry, J. G., Maranas, C. D., Patterson, A. D., and Wood, T. K. (2016) Reversing methanogenesis to capture methane for liquid biofuel precursors, Microb Cell Fact 15, 11. [40] Mayr, S., Latkoczy, C., Kruger, M., Gunther, D., Shima, S., Thauer, R. K., Widdel, F., and Jaun, B. (2008) Structure of an F430 variant from archaea associated with anaerobic oxidation of methane, J. Am. Chem. Soc. 130, 10758-10767. [41] Martin, D., Piera, C., Mazzi, U., Rossin, A., Solans, X., Font-Bardia, M., and Suades, J. (2003) Rhenium and technetium-99m complexes with coenzyme M (MESNA), Dalton T, 30413045. [42] Petrov, A. I., Golovnev, N. N., Dergachev, I. D., and Leshok, A. A. (2013) Complex formation of bismuth(III) with 2-mercaptoethanesulfonic and 3-mercaptopropanesulfonic acids: Experimental and theoretical study, Polyhedron 50, 59-65. [43] Chen, X. e. a. (2016) heavy metals from Kueishantao shallow-sea hydrothermal vents, offshore northeast Taiwan, H, Mar. Syst.? , ? [44] Edgcomb, V. P., Molyneaux, S. J., Saito, M. A., Lloyd, K., Boer, S., Wirsen, C. O., Atkins, M. S., and Teske, A. (2004) Sulfide ameliorates metal toxicity for deep-sea hydrothermal vent archaea, Appl Environ Microbiol 70, 2551-2555.

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Biochemistry

For Table of contents use only D

HS

D

SH

S

O

S O

HS

HS

D

O

5’-dAdo

5’-dAdoD

O

O S

O

O

O

2-mercaptoethanesulfonic acid (HSCoM) HS

HS H

D O S O O

O

?

S

5’-dAdoH 5’-dAdo

D

D

O O


25


Identification and Biosynthesis of 1-Mercaptoethanesulfonic Acid (1

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