Reconstitution of Enzymatic Carbon-Sulfur Bond Formation Reveals

Subscriber access provided by University of Sussex Library

Article

Reconstitution of Enzymatic Carbon-Sulfur Bond Formation Reveals Detoxification-Like Strategy in Fungal Toxin Biosynthesis Daniel H. Scharf, Jan D. Dworschak, Pranatchareeya Chankhamjon, Kirstin Scherlach, Thorsten Heinekamp, Axel A. Brakhage, and Christian Hertweck ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00413 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 4, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

Reconstitution of Enzymatic Carbon-Sulfur Bond Formation Reveals Detoxification-Like Strategy in Fungal Toxin Biosynthesis Daniel H. Scharf,‡,†, # Jan D. Dworschak,γ, # Pranatchareeya Chankhamjon,γ,|| Kirstin Scherlach,γ Thorsten Heinekamp,‡ Axel A. Brakhage,‡,§ and Christian Hertweckγ, §,* AUTHOR ADDRESSES ‡ Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), 07745 Jena, Germany γ Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology (HKI), 07745 Jena, Germany § Friedrich Schiller University Jena, 07743 Jena, Germany

Supporting Information Placeholder ABSTRACT: Gliotoxin is a virulence factor of the

human pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. The activity of this metabolite is mediated by a transannular disulfide bond, a hallmark of the epipolythiodiketopiperazine (ETP) family. Through the creation of fungal gene deletion mutants and heterologous protein expression, we unveiled the critical role of the cytochrome P450 monooxygenase (CYP450) GliC for the stepwise bishydroxylation of the diketopiperazine (DKP) core. We show for the first time the formation of the C-S bond from the DKP in a combined assay of GliC and the glutathione-Stransferase (GST) GliG in vitro. Furthermore, we present experimental evidence for an intermediary imine species. The flexible substrate scope of GliC and GliG in combination parallels P450/GST pairs used in eukaryotic phase I/II detoxification pathways.

Gliotoxin (1) is a potent virulence factor of the opportunistic human pathogenic fungus Aspergillus fumigatus and the prototype of the epipolythiodioxopiperazine (ETP) class of

mycotoxins.1-2 These cyclopeptides are characterized by the presence of an intramolecular disulfide bridge. This particular motif is responsible for the toxicity of the molecule as it inactivates vital proteins and generates reactive oxygen species (ROS) through redox-cycling reaction.3-6 Because of the eminent role of gliotoxin, its biosynthesis has been the subject of many studies.1, 7-9

Figure 1. Two possible reaction pathways for C-S bond formation as early steps in the biosynthesis of 1.

First, the diketopiperazine (DKP) core (2) of gliotoxin is assembled from L-phenylalanine and L-serine by a non-ribosomal peptide synthetase (GliP).10-12 A tentative bishydroxylation of the DKP by the cytochrome P450 (CYP450) monooxygenase GliC may set the basis for the bis-glutathionylation by the glutathione-S-transferase (GST) GliG, yielding the bis-

1 ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 7

glutathione DKP.13-14 A set of enzymes (GliK: γglutamyl cyclotransferase, GliJ: dipeptidase, GliI: C-S bond lyase) catalyzes the degradation of the bisglutathione DKP into the corresponding bis-thiol,15-17 which is transformed into the disulfide by oxidase GliT.18-19 Finally, GliN mediates the amide methylation to generate gliotoxin (Figure 1).14, 20-21 Whereas previous studies indicated that GliC-mediated bishydroxylation precedes glutathione incorporation,13-14, 21-23 the true intermediates, the succession, and the exact mechanism of the reaction have remained elusive.24 Here we report new insight into the reaction intermediates and the mechanism of C-S bond formation catalyzed by an enzyme pair, GliC and GliG. We show that this route, which is highly conserved among all ETP pathways, parallels eukaryotic detoxification pathways. This proposal was corroborated by the successful in vitro biotransformation of diverse synthetic DKPs. In general, two possible avenues are conceivable for the enzymatic C-S bond formation (Figure 1). The first route would proceed via dehydration of a bishydroxylated DKP into an imine intermediate. This intermediate would be used by GliG to catalyze the nucleophilic attack of a thiol glutathione on the iminium ion of the DKP. In the second possible scenario GliG would catalyze glutathione conjugation to the DKP by an SN2 mechanism. To gain further insight into the enzymatic C-S bond formation in gliotoxin biosynthesis we screened all gene deletion mutants for yet unknown intermediates. Thus, we detected a new metabolite (7) with m/z 249.0870 ([M+H]+), which is only formed in trace amounts in the broths of various downstream mutants and of the wild type (wt) (Figure 2C). From ESI-HRMS data the molecular formula C12H13N2O4 of compound (7) was deduced. To elucidate the structure of this compound, sufficient amounts for a full characterization of 7 were isolated from an up-scaled culture (140 L combined culture volume) of a downstream block mutant (∆gliF). Size exclusion, silica gel chromatography and repeated preparative HPLC yielded 1.63 mg of 7. 13C NMR and DEPT 13C spectra of 7 showed signals for two amide carbons, an aromatic ring system, one methine and two additional quaternary carbon atoms. Heteronuclear multiple-bond correlation (HMBC) showed correlations of H-8 to C-9, C-10 and C-14, thus establishing the allocation of the phenylalanine partial structure. The chemical shift of C-3 (81.8 ppm) and the HMBC couplings of 3-OH and H-7 to C3 indicated that the quaternary carbon C-3 is adjacent to an oxygen atom and C-7 (Figure S1).

Figure 2. Metabolic profile of deletion mutants and in vitro reconstitution of GliC activity. A) Metabolic profiles (extracted ion chromatograms) of wild type, ΔgliC, and ∆gliG. B) HPLC-MS monitoring of GliC biotransformation reaction under an 18O2 atmosphere. C) HPLC-MS monitoring of GliC biotransformation reaction. Extracted ion chromatograms from ethyl acetate phase for a) using microsomal fraction without GliC, b) microsomal GliC fraction-mediated transformation of 2. Extracted ion chromatograms from d), e) aqueous phase GliC assay and c) reference compound 7. The structure of 7 with its key HMBC (arrows) and H,HCOSY correlations (bold lines). D) Simulated absorbance spectra of 3, 8, and 9 and the measured absorbance spectrum of the aqueous phase of the GliC assay. E) Formation of a yellow compound in GliC assay and the influence of pH change.

HMBC spectra further showed the correlations of the aromatic protons H-10 and H-14 to C-8 and C-12, thus rigorously determining the structure of 7 (Figure 2C, Table S1). The discovery of 7 provides strong evidence that the DKP hydroxylation occurs in vivo at position C-3. To unequivocally prove the biochemical function of GliC, we set up an enzyme assay. Initial attempts to produce and isolate recombinant GliC from Escherichia coli were unsuccessful, probably due to a hydrophobic Nterminus that is likely to be associated with membranes. Therefore, the enzyme was overproduced in baker's yeast that also produces an essential helper enzyme (cytochrome P450


Page 3 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reductase). The microsomal GliC fraction was isolated and employed in an in vitro assay using DKP 2 as substrate. LC–HRMS monitoring of the enzyme reaction showed that active GliC converts 2 readily into two new compounds, bishydroxylated (5) and monohydroxylated DKP (6). In contrast, no biotransformation of 2 was detected in the assay with the microsomal fraction lacking GliC (Figure 2C). In addition to the in vitro detection of 5 and 6, we also detected small amounts of intermediates by LC–HRMS in the gliG deletion mutant culture extracts (Figure 2C). Both molecules showed the same MSn fragmentation patterns compared to the in vitro assay (Figure S2, S3). Their MSn fragmentation patterns correspond with the sequential loss of two water equivalents for compound 5 (Figure 2A) and the equivalent of one water molecule for compound 6 (Figure S2, S3). These findings corroborate that GliC mediates a DKP bishydroxylation at the α-carbons upstream of GliG. Although 7 is likely a shunt product that results from dehydration of 5 (Figure 2A+C), its structure provides first evidence for the in vivo hydroxylation of C-3. Thus, its discovery supports the model of a stepwise bishydroxylation of the DKP at the αcarbons. Further evidence for the suggested sequence of the reaction was gained by performing the enzyme assay under an 18O2 atmosphere. The subsequent analysis of the products by LC–HRMS showed the production of 6 with the expected mass shift based on the insertion of one molecule of 18O2 (Figure 2B). Furthermore, we uncovered the imine species 3 in the aqueous phase of GliC assay, that is the one of the proposed tautomeric products of dehydration of 5 (Figure S21). We detected two compounds with m/z 249 ([M+H]+) and 231 ([M+H]+) in the aqueous phase of the enzyme assay. HPLC-MS comparisons revealed that the compound with m/z 249 ([M+H]+) is identical with 7 (Figure 2B). Additional evidence for the formation of an imine species is the appearance of an intense yellow color during the GliC enzyme assay, with an absorption maximum at λmax = 436 nm (Figure 2D). Furthermore, the formation of the tautomeric imine species is pH-dependent because of the enlargement (acidic) or reduction (basic) of the conjugated system (Figure 2E, S21). The computational simulation25-28 of the UV/Vis spectra of the tautomers that could derive from dehydration of 5 (Figure S21) showed that the color is likely due to the formation of 3 or 9 (Figure 2D). These data provide further evidence for the formation of distinct species (Figure S21) from 5. Based on these findings, we corroborate a model for C-S bond formation in which GliG mediates the

addition of glutathione downstream of the formation of 3 to form a novel C-S bond. This reaction is a key step in ETP biosynthesis because it installs the sulfur atoms for the bioactive disulfide bridge. The model of C-S bond formation is closely related to a major detoxification pathway in eukaryotes, where phase I is initiated by CYP450-mediated oxygenation, followed by glutathione conjugation in phase II. Owing to this oxygenation-conjugation sequence the hydrophilicity of a xenobiotic substrate is greatly enhanced and its excretion facilitated. It appears that this detoxification pathway has been recruited by fungal secondary metabolism. It is intriguing that the detoxification enzymes have been harnessed to generate a toxin. The high conservation of gliC and gliG orthologues in ETP biosynthesis gene clusters across the fungal kingdom demonstrates the success of this strategy. Notably, the diverse ETP pathways involve DKPs that are built from various amino acids such as tryptophan, phenylalanine, tyrosine, serine, valine, and glycine. Detoxification enzymes typically accept a broad range of substrates. To evaluate the substrate tolerance of GliC, we tested a number of synthetic DKPs29 in biotransformation assays.

Figure 3. Evaluation of the substrate flexibility of GliC and GliG in a coupled assay with various synthetic DKPs. Each bis-glutathione DKP (4, 10b-13b) was detected by HRMS (extracted ion chromatograms) and confirmed by MSn. Therefore, microsomal GliC fractions were incubated with synthetic DKPs (2, 10-13), and the enzyme reaction was monitored by LC–HRMS. The corresponding bishydroxylated products (5, 10a13a) for all substrates could be detected by LC– HRMS (Figure S2, S3, S5, S11–S16). Moreover, all bisglutathione DPKs (4, 10b-13b) could be detected after incubation of the DKPs with a mixture of GliC and GliG (Figure 3, S20). In summary, we have elucidated a key step in the early biosynthesis of



gliotoxin, a potent virulence factor of the human pathogen A. fumigatus. From the large-scale fermentation we isolated an unparalleled monohydroxylated DKP 7, establishing the in vivo hydroxylation at C-3. We also succeeded in producing active GliC and verified through an in vitro assay that the oxygen-dependent enzyme catalyzes a sequential hydroxylation, yielding the corresponding bishydroxylated DKP 5. Moreover, through a series of further analyses we identified molecular oxygen as source for the monohydroxylation. We provide experimental evidence for the downstream formation of an imine 3 species with adjacent incorporation of glutathione. The successful conversion of different DKPs by GliC itself or via a combination assay of GliC and GliG enabled the production of compounds that are hardly accessible through synthetic protocols. Our findings have broader implications because the mechanism and the enzymes involved are highly conserved among all ETP biosynthetic pathways. The most remarkable discovery is that the enzymatic C-S bond formation in ETP biosynthesis mirrors the detoxification pathway of xenobiotics in eukaryotes. Both pathways involve the action of a CYP450 and a GST for the formation of glutathione conjugates (Figure 4). We suggest an evolutionary scenario where detoxification genes were recruited to a biosynthetic pathway to functionalize endogenously produced DKPs. The flexible substrate scope of the bipartite enzyme system reported in this study is in line with its predicted evolutionary origin.

Page 4 of 7

ASSOCIATED CONTENT

The Supporting Information including all experimental details and spectra is available as a pdf free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] Present Addresses †Molecular

and Cellular Physiology, Stanford, CA 94305, USA ||Department of Molecular Biology, Princeton, NJ 08544, USA Author Contributions #These authors contributed equally.

ACKNOWLEDGMENT

We thank A. Perner for MS measurements, M. Steinacker, P. Berthel, J. Schönemann, K.D. Menzel for fermentation, and S. Fricke and C. Schult for technical assistance. Financial support by the National Academy of Sciences (Leopoldina, Grant No. LPDS 2016-05) (for D.H.S.) is gratefully acknowledged. REFERENCES

Figure 4. Overview of P450/GST pairs. A) Biosynthesis of gliotoxin and leukotrienes.30 B) Detoxification pathways for various xenobiotics.31-32

[1] Dolan, S. K., O'Keeffe, G., Jones, G. W., and Doyle, S. (2015) Resistance is not futile: gliotoxin biosynthesis, functionality and utility, Trends Microbiol. 23, 419-428. [2] Welch, T. R., and Williams, R. M. (2014) Epidithiodioxopiperazines. occurrence, synthesis and biogenesis, Nat. Prod. Rep. 31, 1376-1404. [3] Cherblanc, F. L., Lo, Y. P., Herrebout, W. A., Bultinck, P., Rzepa, H. S., and Fuchter, M. J. (2013) Mechanistic and chiroptical studies on the desulfurization of epidithiodioxopiperazines reveal universal retention of configuration at the bridgehead carbon atoms, J. Org. Chem. 78, 11646-11655. [4] Gardiner, D. M., Waring, P., and Howlett, B. J. (2005) The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis, Microbiology 151, 1021-1032. [5] Scharf, D. H., Brakhage, A. A., and Mukherjee, P. K. (2016) Gliotoxin - bane or boon?, Environ. Microbiol. 18, 1096-1109. [6] Scharf, D. H., Heinekamp, T., and Brakhage, A. A. (2014) Human and plant fungal pathogens: the role of secondary metabolites, PLoS Pathog. 10, e1003859. [7] Amatov, T., and Jahn, U. (2014) Gliotoxin: nature's way of making the epidithio bridge, Angew. Chem., Int. Ed. 53, 3312-3314. [8] Scharf, D. H., Heinekamp, T., Remme, N., Hortschansky, P., Brakhage, A. A., and Hertweck, C. (2012) Biosynthesis and function of gliotoxin in Aspergillus fumigatus, Appl. Microbiol. Biotechnol. 93, 467-472. [9] Gardiner, D. M., and Howlett, B. J. (2005) Bioinformatic and expression analysis of the putative gliotoxin biosynthetic gene


Page 5 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology cluster of Aspergillus fumigatus, FEMS Microbiol. Lett. 248, 241248. [10] Sugui, J. A., Pardo, J., Chang, Y. C., Zarember, K. A., Nardone, G., Galvez, E. M., Mullbacher, A., Gallin, J. I., Simon, M. M., and Kwon-Chung, K. J. (2007) Gliotoxin is a virulence factor of Aspergillus fumigatus: gliP deletion attenuates virulence in mice immunosuppressed with hydrocortisone, Eukaryot. Cell 6, 15621569. [11] Kupfahl, C., Heinekamp, T., Geginat, G., Ruppert, T., Hartl, A., Hof, H., and Brakhage, A. A. (2006) Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low-dose mouse infection model, Mol. Microbiol. 62, 292-302. [12] Balibar, C. J., and Walsh, C. T. (2006) GliP, a multimodular nonribosomal peptide synthetase in Aspergillus fumigatus, makes the diketopiperazine scaffold of gliotoxin, Biochemistry 45, 1502915038. [13] Scharf, D. H., Remme, N., Habel, A., Chankhamjon, P., Scherlach, K., Heinekamp, T., Hortschansky, P., Brakhage, A. A., and Hertweck, C. (2011) A dedicated glutathione S-transferase mediates carbon-sulfur bond formation in gliotoxin biosynthesis, J. Am. Chem. Soc. 133, 12322-12325. [14] Davis, C., Carberry, S., Schrettl, M., Singh, I., Stephens, J. C., Barry, S. M., Kavanagh, K., Challis, G. L., Brougham, D., and Doyle, S. (2011) The role of glutathione S-transferase GliG in gliotoxin biosynthesis in Aspergillus fumigatus, Chem. Biol. 18, 542-552. [15] Scharf, D. H., Chankhamjon, P., Scherlach, K., Heinekamp, T., Willing, K., Brakhage, A. A., and Hertweck, C. (2013) Epidithiodiketopiperazine biosynthesis: A four-enzyme cascade converts glutathione conjugates into transannular disulfide bridges, Angew. Chem., Int. Ed. 52, 11092-11095. [16] Scharf, D. H., Chankhamjon, P., Scherlach, K., Heinekamp, T., Roth, M., Brakhage, A. A., and Hertweck, C. (2012) Epidithiol formation by an unprecedented twin carbon-sulfur lyase in the gliotoxin pathway, Angew. Chem., Int. Ed. 51, 10064-10068. [17] Marion, A., Groll, M., Scharf, D. H., Scherlach, K., Glaser, M., Sievers, H., Schuster, M., Hertweck, C., Brakhage, A. A., Antes, I., and Huber, E. M. (2017) Gliotoxin Biosynthesis: Structure, Mechanism, and Metal Promiscuity of Carboxypeptidase GliJ, ACS Chem. Biol. 12, 1874-1882. [18] Scharf, D. H., Remme, N., Heinekamp, T., Hortschansky, P., Brakhage, A. A., and Hertweck, C. (2010) Transannular disulfide formation in gliotoxin biosynthesis and its role in self-resistance of the human pathogen Aspergillus fumigatus, J. Am. Chem. Soc. 132, 10136-10141. [19] Scharf, D. H., Groll, M., Habel, A., Heinekamp, T., Hertweck, C., Brakhage, A. A., and Huber, E. M. (2014) Flavoenzyme-

catalyzed formation of disulfide bonds in natural products, Angew. Chem., Int. Ed. 53, 2221-2224. [20] Scharf, D. H., Habel, A., Heinekamp, T., Brakhage, A. A., and Hertweck, C. (2014) Opposed effects of enzymatic gliotoxin Nand S-methylations, J. Am. Chem. Soc. 136, 11674-11679. [21] Chang, S. L., Chiang, Y. M., Yeh, H. H., Wu, T. K., and Wang, C. C. (2013) Reconstitution of the early steps of gliotoxin biosynthesis in Aspergillus nidulans reveals the role of the monooxygenase GliC, Bioorg. Med. Chem. Lett. 23, 2155-2157. [22] Kim, J., and Movassaghi, M. (2009) Biogenetically inspired syntheses of alkaloid natural products, Chem. Soc. Rev. 38, 30353050. [23] Forseth, R. R., Fox, E. M., Chung, D., Howlett, B. J., Keller, N. P., and Schroeder, F. C. (2011) Identification of cryptic products of the gliotoxin gene cluster using NMR-based comparative metabolomics and a model for gliotoxin biosynthesis, J. Am. Chem. Soc. 133, 9678-9681. [24] Dunbar, K. L., Scharf, D. H., Litomska, A., and Hertweck, C. (2017) Enzymatic carbon-sulfur bond formation in natural product biosynthesis, Chem. Rev. 117, 5521-5577. [25] O'Boyle, N. M., Tenderholt, A. L., and Langner, K. M. (2008) cclib: a library for package-independent computational chemistry algorithms, J. Comput. Chem. 29, 839-845. [26] Perdew, J. P., Emzerhof, M., and Burke, K. (1996) Rationale for mixing exact exchange with density functional approximations, J. Chem. Phys. 105, 9982-9985. [27] Schafer, A., Horn, H., and Ahlrichs, R. (1992) Fully optimized contracted Gaussian-basis sets for atoms Li to Kr, J. Chem. Phys. 97, 2571-2577. [28] Schmidt, M. W., Baldridge, K. K., Boatz, J. A., Elbert, S. T., Gordon, M. S., Jensen, J. H., Koseki, S., Matsunaga, N., Nguyen, K. A., Su, S. J., Windus, T. L., Dupuis, M., and Montgomery, J. A. (1993) General atomic and molecular electronic-structure system, J. Comput. Chem. 14, 1347-1363. [29] Borthwick, A. D. (2012) 2,5-Diketopiperazines: synthesis, reactions, medicinal chemistry, and bioactive natural products, Chem. Rev. 112, 3641-3716. [30] Blair, I. A. (2006) Endogenous glutathione adducts, Curr. Drug Metab. 7, 853-872. [31] Cummins, I., Dixon, D. P., Freitag-Pohl, S., Skipsey, M., and Edwards, R. (2011) Multiple roles for plant glutathione transferases in xenobiotic detoxification, Drug Metab. Rev. 43, 266-280. [32] Dohnal, V., Wu, Q., and Kuca, K. (2014) Metabolism of aflatoxins: key enzymes and interindividual as well as interspecies differences, Arch. Toxicol. 88, 1635-1644.

Insert Table of Contents artwork here



ACS Paragon Plus Environment

Page 6 of 7

6

Page 7 of 7 1 2 3 4 5


ACS Paragon Plus Environment

Reconstitution of Enzymatic Carbon-Sulfur Bond Formation Reveals

Recommend Documents