Identification of an Enzyme Catalyzing the Conversion of

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Identification of a Coenzyme M Synthase: An Enzyme Catalyzing the Conversion of Sulfoacetaldehyde to 2Mercaptoethanesulfonic acid (HSCoM) in Methanogens Robert H White Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00176 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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Biochemistry

Identification of an Enzyme Catalyzing the Conversion of Sulfoacetaldehyde to 2-Mercaptoethanesulfonic acid (HSCoM) in Methanogens Robert H. White* Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States

ABSTRACT: Coenzyme M is an essential coenzyme for the biochemical production of methane. This communication reports on the identification of an enzyme catalyzing the last step in the biosynthesis of coenzyme M in methanogens. Data presented here shows that the enzyme, derived from mj1681, catalyzes the conversion of the aldehyde functional group of sulfoacetaldehyde into the thiol group of HSCoM. This a putative coenzyme M synthase (comF) has sequence similarities with both the MJ0100 and MJ0099 proteins previously shown to be involved in the biosynthesis of homocysteine [Allen, K. D., et. al. (2015) Biochemistry 54, 3129-3132] and both reactions likely proceed by the same mechanism. In the MJ0100 catalyzed reaction, it is proposed [Rauch, B. L., (2017) Biochemistry 56, 1051-1061] that MJ1526 and its homologs in other methanogens likely supply the sulfane sulfur required for the reaction.

Coenzyme M (2-mercaptoethanesulfonic acid, HSCoM) was one of the first coenzymes to be identified in methanogenesis.1 HSCoM is found in all methanogenic archaea, a group of

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microorganisms that are major players in the global carbon cycle, producing ~109 tons of methane annually.2, 3 Despite the fact that most of the reactions/genes involved in HSCoM biosynthesis in methanogens have been identified,4, 5 the one remaining gap in the pathway in the methanogens was the identification of the gene encoding the enzyme catalyzing the final step (Figure 1). It is highly likely that the mechanism for this reaction is analogous to the reaction involved in the biosynthesis of the thiol group of homocysteine in methanogens, where aspartate semialdehyde is converted into homocysteine.6 Two gene products are known to be involved in catalyzing the homocysteine biosynthesis reaction, MJ0100 and MJ0099, with the latter being a ferredoxin II-like protein containing two [4Fe-4S] clusters that supply the electrons required for the conversion of the aldehyde group of sulfoacetaldehyde into a thiol. The discovery of this reaction represented a previously unknown biochemical route for the formation of thiols. Here, genes associated with the genes encoding the enzymes known to be involved in HSCoM biosynthesis were analyzed in order to identify the enzyme catalyzing the final step of HSCoM biosynthesis. One clear connection was observed with the Methanocaldococcus jannaschii gene, mj1681 and its homologs, which have a unknown function(s) and are only found in the methanogens. All of these proteins contain two conserved cysteines—one at C72 and the other at C172 (M. jannaschii numbering) in the N-terminal portion of the protein (Figure 2). MJ1681 also has homology with the N-terminal portion of MJ0100 up to residue ~275. The remaining portion of MJ1681 has a sequence containing two ferredoxin domains (CX2CX2CX5C and CX2CX2CX2C) in the C-terminus of the enzyme that replace two ferredoxin

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Biochemistry

domains (CX2CX2CX3C and CX2CX2CX3C) present in MJ0099, which is required for homocysteine biosynthesis. These ferredoxin clusters likely conduct electrons into the active site of MJ1681. Currently, no X-ray structure exists for either MJ0100 or MJ1681 or any of their homologs. In order to test the proposal that MJ1681 is the enzyme catalyzing the final step of HSCoM biosynthesis in the methanogens, the gene was cloned into pT7-7 for recombinant expression in E. coli. The recombinant cells contained abundant protein as measured by SDSPAGE analysis (Figure S1) based on the intensity of the band observed, which had a measured mass of 42 kD. That this band contained the desired protein was confirmed by sequencing the band by MALDI. The introduction of 0.125 mM Fe(II), 0.125 mM Fe(II) plus 2 mM mercaptoethanol, or 0.125 mM Fe(II) plus 10 mM sulfoacetaldehyde to the growth media did not alter the amount of MJ1681 protein expressed (Figure S1, lanes 3-5). Soluble cell extracts obtained by sonication of the cells followed by centrifugation were found to be devoid of MJ1681 protein even after heating the cell extract over a range of temperatures from 50 to 80 oC. The MJ1681 protein was only identified and confirmed by MALDI in the cell pellets. All gel band(s) considered to contain MJ1681 were assayed by MALDI-MS and, when the desired protein was detected, five tryptic peptides with the desired sequences expected for MJ1681 were identified. In other cases, only E. coli proteins were identified. Attempts to produce soluble protein by sonicating the cell pellets in the presence of trehalose 7 also did not result in soluble enzyme being detected by SDS-PAGE analyses followed by MALDIMS analysis of the bands observed where the known protein moves in the gel. Since M. jannaschii does not currently have a genetic system that allows one to generate a gene knock-out of the MJ1681 gene. A knock-out of the homologous gene in M.

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maripaludis, Mmp1603, was generated. Growth of the resulting cells had no dependence the presence or absence of HSCoM added to the media (personal communication of Thomas Lee, University of Washington). I concluded that this observation can be explained as a result in the presence of one or more additional enzyme(s) in these cells that are also able to catalyze this last step in HSCoM biosynthesis. The thiol group in coenzyme B, which is likely produced by a similar reaction, 4 is a possible candidate. The most likely enzyme to do the reaction would be the homocysteine synthetase which is present in all methanogens, that catalyzed the same basic reaction. Alternately another enzyme catalyzing the biosynthesis, yet to be identified, could be catalyzing the reaction. HSCoM was measured directly from E. coli cells expressing MJ1681, which produced HSCoM when grown in the presence of sulfoacetaldehyde. The concentration of the HSCoM in the cells was ~1 mM, assuming that the intercellular cell volume was 50% of the measured dry weight. This calculated concentration was 300 x higher than that measured in the media. No HSCoM was found in the media or in the cells of E. coli containing the plasmid without the mj1681 gene. These observations demonstrated that the expression of MJ1681 in E. coli allowed the cells to produce HSCoM when grown in the presence of sulfoacetaldehyde, indicating that a system(s) already present in E. coli 8 was able to transport sulfoacetaldehyde into the cells and supply the required sulfane sulfur and electrons to the enzyme to allow the cells to produce HSCoM. Using the MJ1681 numbering, the first two conserved cysteines in MJ1681 correspond to C72 and C166 (Figure. 2). This is followed by two [4Fe-4S] ferredoxin motifs: the first at C296, C299, C302, and C308 and the second at C326, C329, C332, and C335 (Figure 3). These

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Biochemistry

cysteines are clearly analogous to the 4Fe-4S ferredoxin clusters present in a Clostridium acidurici ferredoxin and dihydropyrimidine dehydrogenase (DHPDH). 9 These ferredoxin clusters are likely used to transfer electrons into the active site of the enzyme for reduction of the enzymatically generated disulfides (Fig. 4, steps 4 and 7). The C-terminus of the MJ1681 with the conserved ferredoxin domains, has a structure like the C-terminus of (DHPDH) that also contains ferredoxin domains. 10-12 As seen with DHPDH, this suggests that one of the two ferredoxin domains is the site for the introduction of the required electrons for thiol formation into the enzyme. In DHPDH, NADH transfers electrons to FAD, which then transfers one electron at a time into nFeS2A then to nFeS1A and, after electron transfer through two more clusters, to an acceptor FMN. The H2FMN then does a two-electron reduction of the pyrimidine substrate. We propose that in our coenzyme M synthase this second FMN is not present and the nFeS1A like cluster then reduces the disulfide bond between cys-1 and cys-2 in our mechanism by two single electron transfers (Figure 4). At this point we have no way to distinguish cys-1 from cys-2. The homolog of MJ0100 in Methanosarcina acetovorans is MA1821, which has only two conserved cysteines, C54 and C131. These cysteines do not correspond to the positions of the conserved C54 and C131 in MJ1681. The C131A variant in MA1821 was able to grow on sulfideonly medium, whereas the C54A variant in MA1821 was not able to grow under the same conditions. 13 These observations indicate that the first cysteine in MA1821 is critical for catalysis. We propose that this is the position of the persulfide. Neither of these cysteines is close to the MJ1681 C72 and C166 in the alignment of the two proteins (Figure 2). Thus, either the enzymes really have the same structures and the cysteines are in different positions in the

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sequence and/or the proteins have different structures but form a similar active site that involves these cysteines. An excellent model for the reduction of the disulfides in our proposed mechanism (Figure 4) can be found in ferredoxin:thioredoxin reductase (FTR) found in plants.14 Here, a reduced [Fe2S2] ferredoxin donates single electrons to FTR containing an active site comprised of an [Fe4S+2 cluster and an adjacent disulfide. After the disulfide is reduced with two electrons, the resulting thiols then reduce the thioredoxin disulfide. We propose that in our case, the electrons are used to reduce the required disulfides generated in our proposed mechanism. This is similar to that observed in the heterodisulfide reductase that does not require a flavin. 15 It appears that MJ1681 has homology to members of the FTR protein family related to the plant enzyme. 16 Based on the above observations, we propose that the first step in the enzymatic reaction is the transfer of a persulfide sulfur from MJ1526 to one of the two conserved cysteines in the N-terminus of MJ1681 (Figure 4). Since there is no way, at this time, to determine the true acceptor thiol, they will be called cys-1 and cys-2. The thiol of the persulfide at cys-1 then adds to the aldehyde of sulfoacetaldehyde in step 2 to form a thiohemialdehyde. Although no such reactions with persulfides appear to have been described, such reactions with thiols are well-documented.17, 18 A thiolate anion at cys-2 then attacks the disulfide between cys-1 with cleavage of the disulfide bond and elimination of the protonated alcohol as water, leading to the formation of a thioaldehyde adduct of sulfoacetaldehyde (step 3). It is well established, that thiol aldehyde are not expected to be stable. 19 In step 4, two single electron transfers from the ferredoxins reduce the disulfide bond formed between cys-1 and cys-2. The

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Biochemistry

resulting cys-2 thiolate then does a nucleophilic attack on the sulfur of the bound thioaldehyde in step 5. The most significant difference between the reaction of thiocarbonyl and carbonyl chemistry is that nucleophiles do not always attack the carbon but can sometimes attack the sulfur instead of the carbon.19 This is an important aspect of my reaction scheme, where the sulfur is a nucleophile at the thiocarbonyl sulfur. This reaction is essentially carrying out a reduction of the thiocarbonyl. After a disulfide bond migration in step 6, HSCoM is released and a disulfide is formed between cys-1 and cys-2. Finally, in a second two-electron reduction, the disulfide is reduced in step 7. Insight into the source of the sulfur can be gained from recent work demonstrating that MA1821 in Methanosarcina acetivorans supplies persulfide for both cysteine and homocysteine biosynthesis in this methanogen, 20 which is expected to be a model for sulfane transfer in the methanogens. The sulfane sulfur-donating enzyme is present in all methanogens, and the homolog in M. jannaschii, MJ1526, is likely supplying the sulfane sulfur required by MJ1681 to produce HSCoM. Such sulfur mobilization routes are widespread in prokaryote and eukaryotes. 21

ASSOCIATED CONTENT Supported information The supporting information contains materials, methods, and one additional figure (PDF).

Accession Codes MJ1681, Q59074. AUTHOR INFORMATION

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Corresponding author *E-mail: [email protected]. Phone: 540-231-6605. Fax: 540-231-9070. Present Address Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 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 MALDI experiments. The mass spectrometry resources 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 Valerie Cash for preparing the recombinant E. coli cells, growing the cells and preparing Figures 2 and 3. I would also like to thank Walter Niehaus, Janet Webster and Kylie Allen for help in editing the manuscript. ABBREVIATIONS TCEP; tris-(2-carboxyethyl)phosphine hydrochloride ; monobromobimane, B. REFERENCES [1] McBride, B. C., and Wolfe, R. S. (1971) A new coenzyme of methyl transfer, coenzyme M, Biochemistry 10, 2317-2324. [2] Thauer, R. K., Kaster, A. K., Seedorf, H., Buckel, W., and Hedderich, R. (2008) Methanogenic archaea: ecologically relevant differences in energy conservation, Nature reviews. Microbiology 6, 579-591.

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Biochemistry

[3] Ferry, J. G. (1997) Methane: small molecule, big impact, Science 278, 1413-1414. [4] Grochowski, L. L., and White, R. H. (2010) Biosynthesis of the Methanogenic coenzymes, In Comprehensive Natural Products II: Chemistry and Biology (Begley, T. P., Ed.), pp 711-748, Elsevier Ltd, New York. [5] 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. [6] 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. [7] Leibly, D. J., Nguyen, T. N., Kao, L. T., Hewitt, S. N., Barrett, L. K., and Van Voorhis, W. C. (2012) Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins, PloS one 7, e52482. [8] van der Ploeg, J. R., Eichhorn, E., and Leisinger, T. (2001) Sulfonate-sulfur metabolism and its regulation in Escherichia coli, Arch Microbiol 176, 1-8. [9] Duee, E. D., Fanchon, E., Vicat, J., Sieker, L. C., Meyer, J., and Moulis, J. M. (1994) Refined crystal structure of the 2[4Fe-4S] ferredoxin from Clostridium acidurici at 1.84 A resolution, J Mol Biol 243, 683-695. [10] Dobritzsch, D., Schneider, G., Schnackerz, K. D., and Lindqvist, Y. (2001) Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anticancer drug 5-fluorouracil, EMBO J 20, 650-660. [11] Dobritzsch, D., Ricagno, S., Schneider, G., Schnackerz, K. D., and Lindqvist, Y. (2002) Crystal structure of the productive ternary complex of dihydropyrimidine dehydrogenase with NADPH and 5iodouracil. Implications for mechanism of inhibition and electron transfer, J Biol Chem 277, 1315513166. [12] Lohkamp, B., Voevodskaya, N., Lindqvist, Y., and Dobritzsch, D. (2010) Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination, Biochim Biophys Acta 1804, 2198-2206. [13] Rauch, B. J., Gustafson, A., and Perona, J. J. (2014) Novel proteins for homocysteine biosynthesis in anaerobic microorganisms, Mol Microbiol 94, 1330-1342. [14] Walters, E. M., Garcia-Serres, R., Jameson, G. N., Glauser, D. A., Bourquin, F., Manieri, W., Schurmann, P., Johnson, M. K., and Huynh, B. H. (2005) Spectroscopic characterization of site-specific [Fe(4)S(4)] cluster chemistry in ferredoxin:thioredoxin reductase: implications for the catalytic mechanism, J Am Chem Soc 127, 9612-9624. [15] Kunkel, A., Vaupel, M., Heim, S., Thauer, R. K., and Hedderich, R. (1997) Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri is not a flavoenzyme, Eur J Biochem 244, 226234. [16] Kumar, A. K., Kumar, R. S., Yennawar, N. H., Yennawar, H. P., and Ferry, J. G. (2015) Structural and Biochemical Characterization of a Ferredoxin:Thioredoxin Reductase-like Enzyme from Methanosarcina acetivorans, Biochemistry 54, 3122-3128. [17] Gunshore, S., Brush, E. J., and Hamilton, G. A. (1985) Equilibrium-Constants for the Formation of Glyoxylate Thiohemiacetals and Kinetic Constants for Their Oxidation by O-2 Catalyzed by LHydroxy Acid Oxidase, Bioorganic chemistry 13, 1-13. [18] Cleary, J. A., Doherty, W., Evans, P., and Malthouse, J. P. (2015) Quantifying tetrahedral adduct formation and stabilization in the cysteine and the serine proteases, Biochim Biophys Acta 1854, 1382-1391. [19] Mcgregor, W. M., and Sherrington, D. C. (1993) Some Recent Synthetic Routes to Thioketones and Thioaldehydes, Chemical Society Reviews 22, 199-204.

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[20] Rauch, B. J., Klimek, J., David, L., and Perona, J. J. (2017) Persulfide Formation Mediates Cysteine and Homocysteine Biosynthesis in Methanosarcina acetivorans, Biochemistry 56, 1051-1061. [21] Leimkuhler, S., Buhning, M., and Beilschmidt, L. (2017) Shared Sulfur Mobilization Routes for tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Prokaryotes and Eukaryotes, Biomolecules 7.

Figure 1. Pathway for HSCoM biosynthesis in the methanogens. Pathway A is found in the class I methanogens and pathway B is found in class II methanogens. 6

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Biochemistry

* * * *

Figure 2. Alignment of Mj1681 and Mj0100. This figure shows homology at the N’-terminus of these two proteins and location of two cysteine residues shown to be conserved in each strain (Mj1681 shown with red * and Mj0100 shown with blue *). Blue lines show two CBS domains in Mj0100 (CBS1 aa392-445, CBS2 aa458-506) known to bind SAM and S-methyl-5’-thioadenosine.

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*

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* * *

*

Figure 3. Alignment of Mj1681 with FdxA from Clostridium acidurici, and DHPDH from Homo sapiens. The figure shows the alignment of the two [4Fe-4S] ferredoxin motifs at the C’terminus of Mj1681 with the Ca ferredoxin A, and the similarities of the Mj1681 C’-terminus with the two ferredoxin domains at the C-terminus of dihydropyrimidine dehydrogenase (DHPDH).

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Biochemistry

H+

H+ O

O3S H2C

C HS

OH

O 3S H2C

H S

2

SH

C

H+ S

H S

S

MJ1526-SH

1

MJ1526-SSH

3

sulfane donor SH

HS cys-1

O 3S

cys-2

H

H2C

C S

MJ1681 [4Fe-4S]+1

2x

e-

S

[4Fe-4S]+2

cys-1

7 [4Fe-4S]+2

H2O

S cys-2

2 H+

[4Fe-4S]+1

H+ O3S

S

S

H+

H2C

C

H

[4Fe-4S]+1

[4Fe-4S]+1

[4Fe-4S]+2

[4Fe-4S]+2

4 2 x e-

S H

O 3S H2C

O3S H2C

CH2 SH

HS

6

H

C

H+

S H+

S

S

S

5

Figure 4. Proposed mechanism for the enzymatic conversion of sulfoacetaldehyde to HSCoM.

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For Table of contents use only

HSCoM synthase O3S H2C

O

H2O O3S CH2 CH2SH

C

H sulfoacetaldehyde

[S]o + 4H+ + 4e-

coenzyme M

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Biochemistry

A

HSO3

O O

ComA

H 2O

O O3S

O

Pi

O O3S

ComB

OP

OP

O OH

PEP

NAD+ ComC

B O PO

O

Pi O

NH3

+

MA3297

O NH3

O

HSO3

+

MA3297

O3S

O

AspAT O

NH3

NADH + H+

O3S

+

O O

H+

O3S

SH

Coenzyme M

CO2

[S]o + 4H+ + 4eComF

H

O3S O


ComDE

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Biochemistry

* * *

*

* * *


*

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H+

H+ O

O3S H 2C

C HS

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OH

O3S H 2C

H S

H+

2

SH

C S

H S

S

MJ1526-SH

1

MJ1526-SSH

3

sulfane donor SH

HS cys-1

O3S

cys-2

H

H 2C

C S

MJ1681 [4Fe-4S]+1

2 x e-

S

[4Fe-4S]+2

cys-1

7 [4Fe-4S]+2

H 2O

S cys-2

2 H+

[4Fe-4S]+1

H+ O3S

S

S

H+

H 2C

C

H

S H

O3S H 2C

O3S H 2C

CH2 SH

HS

6

H

C

H+

S H+

S

S

5


S

[4Fe-4S]+1

[4Fe-4S]+1

[4Fe-4S]+2

[4Fe-4S]+2

4 2 x e-

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Biochemistry

HSCoM synthase O3S H 2C

O

H 2O O3S CH2 CH2SH

C

H sulfoacetaldehyde

[S]o + 4H+ + 4e-

coenzyme M


Identification of an Enzyme Catalyzing the Conversion of

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