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ABSTRACT Subtilosin A is a 35-residue, ribosomally synthesized bacteriocin encoded by the _sbo_-_alb_ operon of _Bacillus subtilis_. It is composed of a head-to-tail circular peptide

backbone that is additionally restrained by three unusual thioether bonds between three cysteines and the α-carbon of one threonine and two phenylalanines, respectively. In this study, we

demonstrate that these bonds are synthesized by the radical _S_-adenosylmethionine enzyme AlbA, which is encoded by the _sbo_-_alb_ operon and comprises two [4Fe-4S] clusters. One [4Fe-4S]

cluster is coordinated by the prototypical CXXXCXXC motif and is responsible for the observed _S_-adenosylmethionine cleavage reaction, whereas the second [4Fe-4S] cluster is required for

the generation of all three thioether linkages. On the basis of the obtained results, we propose a new radical mechanism for thioether bond formation. In addition, we show that AlbA-directed

substrate transformation is leader-peptide dependent, suggesting that thioether bond formation is the first step during subtilosin A maturation. Access through your institution Buy or

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HISTORY * _ 16 APRIL 2012 In the version of this article initially published, _S_-adenosyl methionine was drawn with a radical instead of a cation in Figure 4. The error has been corrected

in the HTML and PDF versions of the article. _ REFERENCES * Hallen, H.E., Luo, H., Scott-Craig, J.S. & Walton, J.D. Gene family encoding the major toxins of lethal _Amanita_ mushrooms.

_Proc. Natl. Acad. Sci. USA_ 104, 19097–19101 (2007). Article  CAS  PubMed  Google Scholar  * Cascales, L. & Craik, D.J. Naturally occurring circular proteins: distribution, biosynthesis

and evolution. _Org. Biomol. Chem._ 8, 5035–5047 (2010). Article  CAS  PubMed  Google Scholar  * Maqueda, M. et al. Genetic features of circular bacteriocins produced by Gram-positive

bacteria. _FEMS Microbiol. Rev._ 32, 2–22 (2008). Article  CAS  PubMed  Google Scholar  * Babasaki, K., Takao, T., Shimonishi, Y. & Kurahashi, K. Subtilosin A, a new antibiotic peptide

produced by _Bacillus subtilis_ 168: isolation, structural analysis, and biogenesis. _J. Biochem._ 98, 585–603 (1985). Article  CAS  PubMed  Google Scholar  * Shelburne, C.E. et al. The

spectrum of antimicrobial activity of the bacteriocin subtilosin A. _J. Antimicrob. Chemother._ 59, 297–300 (2007). Article  CAS  PubMed  Google Scholar  * Sutyak, K.E. et al. Spermicidal

activity of the safe natural antimicrobial peptide subtilosin. _Infect. Dis. Obstet. Gynecol._ 2008, 540758 (2008). Article  PubMed  Google Scholar  * Silkin, L., Hamza, S., Kaufman, S.,

Cobb, S.L. & Vederas, J.C. Spermicidal bacteriocins: lacticin 3147 and subtilosin A. _Bioorg. Med. Chem. Lett._ 18, 3103–3106 (2008). Article  CAS  PubMed  Google Scholar  * Huang, T. et

al. Isolation of a variant of subtilosin A with hemolytic activity. _J. Bacteriol._ 191, 5690–5696 (2009). Article  CAS  PubMed  Google Scholar  * Kawulka, K. et al. Structure of subtilosin

A, an antimicrobial peptide from _Bacillus subtilis_ with unusual posttranslational modifications linking cysteine sulfurs to α-carbons of phenylalanine and threonine. _J. Am. Chem. Soc._

125, 4726–4727 (2003). Article  CAS  PubMed  Google Scholar  * Kawulka, K.E. et al. Structure of subtilosin A, a cyclic antimicrobial peptide from _Bacillus subtilis_ with unusual sulfur to

α-carbon cross-links: formation and reduction of α-thio-α-amino acid derivatives. _Biochemistry_ 43, 3385–3395 (2004). Article  CAS  PubMed  Google Scholar  * Liu, W.-T. et al. Imaging mass

spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of _Bacillus subtilis_. _Proc. Natl. Acad. Sci. USA_ 107, 16286–16290 (2010). Article  CAS  PubMed  Google

Scholar  * Lee, H., Churey, J.J. & Worobo, R.W. Biosynthesis and transcriptional analysis of thurincin H, a tandem repeated bacteriocin genetic locus, produced by _Bacillus

thuringiensis_ SF361. _FEMS Microbiol. Lett._ 299, 205–213 (2009). Article  CAS  PubMed  Google Scholar  * Sit, C.S., van Belkum, M.J., McKay, R.T., Worobo, R.W. & Vederas, J.C. The 3D

solution structure of thurincin H, a cacteriocin with four sulfur to α-carbon crosslinks. _Angew. Chem. Int. Edn Engl._ 50, 8718–8721 (2011). Article  CAS  Google Scholar  * Rea, M.C. et al.

Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against _Clostridium difficile_. _Proc. Natl. Acad. Sci. USA_ 107, 9352–9357 (2010). Article  CAS

  PubMed  Google Scholar  * Sit, C.S., McKay, R.T., Hill, C., Ross, R.P. & Vederas, J.C. The 3D structure of thuricin CD, a two-component bacteriocin with cysteine sulfur to α-carbon

cross-links. _J. Am. Chem. Soc._ 133, 7680–7683 (2011). Article  CAS  PubMed  Google Scholar  * Zheng, G., Yan, L.Z., Vederas, J.C. & Zuber, P. Genes of the _sbo_-_alb_ locus of

_Bacillus subtilis_ are required for production of the antilisterial bacteriocin subtilosin. _J. Bacteriol._ 181, 7346–7355 (1999). CAS  PubMed Central  PubMed  Google Scholar  * Zheng, G.,

Hehn, R. & Zuber, P. Mutational analysis of the _sbo_-_alb_ locus of _Bacillus subtilis_: identification of genes required for subtilosin production and immunity. _J. Bacteriol._ 182,

3266–3273 (2000). Article  CAS  PubMed  Google Scholar  * Nakano, M.M., Zheng, G. & Zuber, P. Dual control of _sbo_-_alb_ operon expression by the Spo0 and ResDE systems of signal

transduction under anaerobic conditions in _Bacillus subtilis_. _J. Bacteriol._ 182, 3274–3277 (2000). Article  CAS  PubMed  Google Scholar  * Albano, M. et al. The Rok protein of _Bacillus

subtilis_ represses genes for cell surface and extracellular functions. _J. Bacteriol._ 187, 2010–2019 (2005). Article  CAS  PubMed  Google Scholar  * González-Pastor, J.E., Hobbs, E.C.

& Losick, R. Cannibalism by sporulating bacteria. _Science_ 301, 510–513 (2003). Article  PubMed  Google Scholar  * Sofia, H.J., Chen, G., Hetzler, B., Reyes-Spindola, J. & Miller,

N. Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information

visualization methods. _Nucleic Acids Res._ 29, 1097–1106 (2001). Article  CAS  PubMed  Google Scholar  * Frey, P.A., Hegeman, A.D. & Ruzicka, F.J. The radical SAM superfamily. _Crit.

Rev. Biochem. Mol. Biol._ 43, 63–88 (2008). Article  CAS  PubMed  Google Scholar  * Duschene, K.S., Veneziano, S.E., Silver, S.C. & Broderick, J.B. Control of radical chemistry in the

AdoMet radical enzymes. _Curr. Opin. Chem. Biol._ 13, 74–83 (2009). Article  CAS  PubMed  Google Scholar  * Chirpich, T.P., Zappia, V., Costilow, R.N. & Barker, H.A. Lysine

2,3-aminomutase. Purification and properties of a pyridoxal phosphate and S-adenosylmethionine-activated enzyme. _J. Biol. Chem._ 245, 1778–1789 (1970). CAS  PubMed  Google Scholar  * Layer,

G. et al. Structural and functional comparison of HemN to other radical SAM enzymes. _Biol. Chem._ 386, 971–980 (2005). Article  CAS  PubMed  Google Scholar  * Guianvarc'h, D.,

Florentin, D., Tse Sum Bui, B., Nunzi, F. & Marquet, A. Biotin synthase, a new member of the family of enzymes which uses S-adenosylmethionine as a source of deoxyadenosyl radical.

_Biochem. Biophys. Res. Commun._ 236, 402–406 (1997). Article  CAS  PubMed  Google Scholar  * Ugulava, N.B., Gibney, B.R. & Jarrett, J.T. Biotin synthase contains two distinct

iron-sulfur cluster binding sites: chemical and spectroelectrochemical analysis of iron-sulfur cluster interconversions. _Biochemistry_ 40, 8343–8351 (2001). Article  CAS  PubMed  Google

Scholar  * Berkovitch, F. Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. _Science_ 303, 76–79 (2004). Article  CAS  PubMed  Google Scholar  * Miller,

J.R. et al. _Escherichia coli_ LipA is a lipoyl synthase: _in vitro_ biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein. _Biochemistry_ 39,

15166–15178 (2000). Article  CAS  PubMed  Google Scholar  * Cicchillo, R.M. et al. Lipoyl synthase requires two equivalents of _S_-adenosyl-L-methionine to synthesize one equivalent of

lipoic acid. _Biochemistry_ 43, 6378–6386 (2004). Article  CAS  PubMed  Google Scholar  * Santamaria-Araujo, J.A. et al. The tetrahydropyranopterin structure of the sulfur-free and

metal-free molybdenum cofactor precursor. _J. Biol. Chem._ 279, 15994–15999 (2004). Article  CAS  PubMed  Google Scholar  * Hänzelmann, P. & Schindelin, H. Binding of 5′-GTP to the

C-terminal FeS cluster of the radical S-adenosylmethionine enzyme MoaA provides insights into its mechanism. _Proc. Natl. Acad. Sci. USA_ 103, 6829–6834 (2006). Article  PubMed  Google

Scholar  * Pierrel, F., Douki, T., Fontecave, M. & Atta, M. MiaB protein is a bifunctional radical-S-adenosylmethionine enzyme involved in thiolation and methylation of tRNA. _J. Biol.

Chem._ 279, 47555–47563 (2004). Article  CAS  PubMed  Google Scholar  * Hernández, H.L. et al. MiaB, a bifunctional radical-_S_-adenosylmethionine enzyme involved in the thiolation and

methylation of tRNA, contains two essential [4Fe-4S] clusters. _Biochemistry_ 46, 5140–5147 (2007). Article  PubMed  Google Scholar  * Lee, K.-H. et al. Characterization of RimO, a new

member of the methylthiotransferase subclass of the radical SAM superfamily. _Biochemistry_ 48, 10162–10174 (2009). Article  CAS  PubMed  Google Scholar  * Yokoyama, K., Numakura, M., Kudo,

F., Ohmori, D. & Eguchi, T. Characterization and mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin. _J. Am. Chem. Soc._ 129, 15147–15155 (2007). Article 

CAS  PubMed  Google Scholar  * Grove, T.L., Ahlum, J.H., Sharma, P., Krebs, C. & Booker, S.J. A consensus mechanism for radical SAM-dependent dehydrogenation? BtrN contains two [4Fe-4S]

clusters. _Biochemistry_ 49, 3783–3785 (2010). Article  CAS  PubMed  Google Scholar  * Fang, Q., Peng, J. & Dierks, T. Post-translational formylglycine modification of bacterial

sulfatases by the radical S-adenosylmethionine protein AtsB. _J. Biol. Chem._ 279, 14570–14578 (2004). Article  CAS  PubMed  Google Scholar  * Grove, T.L., Lee, K.-H., St. Clair, J., Krebs,

C. & Booker, S.J. In vitro characterization of AtsB, a radical SAM formylglycine-generating enzyme that contains three [4Fe-4S] clusters. _Biochemistry_ 47, 7523–7538 (2008). Article 

CAS  PubMed  Google Scholar  * Hagen, K. & Watson, A. Synthetic routes to iron sulfide (Fe2S2, Fe3S4, Fe4S4, and Fe6S9), clusters from the common precursor

tetrakis(ethanethiolate)ferrate2– ion ([Fe(SC2H5)4]2–): structures and properties of [Fe3S4(SR)4]3– and bis(ethanethiolate)nonathioxohexaferrate4– ion ([Fe6S9(SC2H5)2]4–), examples of the

newest types of Fe-S-SR clusters. _J. Am. Chem. Soc._ 105, 3905–3913 (1983). Article  CAS  Google Scholar  * Külzer, R., Pils, T., Kappl, R., Hüttermann, J. & Knappe, J. Reconstitution

and characterization of the polynuclear iron-sulfur cluster in pyruvate formate-lyase-activating enzyme. Molecular properties of the holoenzyme form. _J. Biol. Chem._ 273, 4897–4903 (1998).

Article  PubMed  Google Scholar  * Ugulava, N.B., Gibney, B.R. & Jarrett, J.T. Iron-sulfur cluster interconversions in biotin synthase: dissociation and reassociation of iron during

conversion of [2Fe-2S] to [4Fe-4S] clusters. _Biochemistry_ 39, 5206–5214 (2000). Article  CAS  PubMed  Google Scholar  * Duschene, K.S. & Broderick, J.B. The antiviral protein viperin

is a radical SAM enzyme. _FEBS Lett._ 584, 1263–1267 (2010). Article  CAS  PubMed  Google Scholar  * Chatterjee, A. et al. Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands

the radical SAM superfamily. _Nat. Chem. Biol._ 4, 758–765 (2008). Article  CAS  PubMed  Google Scholar  * Murphy, K. et al. Genome mining for radical SAM protein determinants reveals

multiple sactibiotic-like gene clusters. _PLoS ONE_ 6, e20852 (2011). Article  CAS  PubMed  Google Scholar  * Oman, T.J. & van der Donk, W.A. Follow the leader: the use of leader

peptides to guide natural product biosynthesis. _Nat. Chem. Biol._ 6, 9–18 (2010). Article  CAS  PubMed  Google Scholar  * Xie, L., Miller, L., Chatterjee, C. & Averin, O. Lacticin 481:

_in vitro_ reconstitution of lantibiotic synthetase activity. _Science_ 303, 679–681 (2004). Article  CAS  PubMed  Google Scholar  * Chatterjee, C., Paul, M., Xie, L. & van der Donk,

W.A. Biosynthesis and mode of action of lantibiotics. _Chem. Rev._ 105, 633–684 (2005). Article  CAS  PubMed  Google Scholar  * Roach, P.L., Clifton, I., Hensgens, C. & Shibata, N.

Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. _Nature_ 387, 827–830 (1997). Article  CAS  PubMed  Google Scholar  * Layer, G.,

Verfürth, K., Mahlitz, E. & Jahn, D. Oxygen-independent coproporphyrinogen-III oxidase HemN from _Escherichia coli_. _J. Biol. Chem._ 277, 34136–34142 (2002). Article  CAS  PubMed 

Google Scholar  Download references ACKNOWLEDGEMENTS We would like to thank N. Fritzler and J. Bamberger for practical realization of the HPLC-HRMS measurements. In addition, we would like

to thank the whole Marahiel group for fruitful discussions. Furthermore, we gratefully acknowledge financial support from the Deutsche Forschungsgemeinschaft and from the LOEWE Center for

Synthetic Microbiology. AUTHOR INFORMATION Author notes * Michael J Gattner Present address: Present address: Fakultät für Chemie und Pharmazie, Ludwig-Maximilians-Universität München,

München, Germany., AUTHORS AND AFFILIATIONS * Department of Chemistry–Biochemistry, Philipps-Universität Marburg, Marburg, Germany Leif Flühe, Thomas A Knappe, Michael J Gattner, Antje

Schäfer, Olaf Burghaus, Uwe Linne & Mohamed A Marahiel Authors * Leif Flühe View author publications You can also search for this author inPubMed Google Scholar * Thomas A Knappe View

author publications You can also search for this author inPubMed Google Scholar * Michael J Gattner View author publications You can also search for this author inPubMed Google Scholar *

Antje Schäfer View author publications You can also search for this author inPubMed Google Scholar * Olaf Burghaus View author publications You can also search for this author inPubMed 

Google Scholar * Uwe Linne View author publications You can also search for this author inPubMed Google Scholar * Mohamed A Marahiel View author publications You can also search for this

author inPubMed Google Scholar CONTRIBUTIONS T.A.K. and M.A.M. initiated the project. M.J.G., L.F. and T.A.K. conceived the experiments. M.J.G. performed preliminary experiments for the

characterization of AlbA. L.F. performed most experiments. M.J.G. and L.F. prepared the figures. O.B. carried out the EPR measurements. U.L. designed the HPLC-MS gradients and carried out

the measurements. A.S. cloned, expressed and purified AlbA. L.F. and T.A.K. analyzed and interpreted the obtained data. L.F., T.A.K. and M.A.M. coordinated the project and wrote the

manuscript. CORRESPONDING AUTHOR Correspondence to Mohamed A Marahiel. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY TEXT AND FIGURES Supplementary Methods and Supplementary Results (PDF 11619 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Flühe, L.,

Knappe, T., Gattner, M. _et al._ The radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A. _Nat Chem Biol_ 8, 350–357 (2012). https://doi.org/10.1038/nchembio.798

Download citation * Received: 22 June 2011 * Accepted: 22 December 2011 * Published: 26 February 2012 * Issue Date: April 2012 * DOI: https://doi.org/10.1038/nchembio.798 SHARE THIS ARTICLE

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