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news & views fibrils, not just the ends). Somehow the lipids control fibril growth, blocking sideways extension and preventing limitless extension of the growing ends—this remains a rather mysterious phenomenon. Thus although the vesicles strongly attract αSN monomers, at most one fibril is allowed to form per vesicle, for reasons as yet not understood and consequently not modeled by Galvagnion et al. Modeled this way, the rate of fibrillation does not depend on free αSN concentration, unlike for fibrillation in solution; the trick is the high degree of binding to vesicles—in fact, vesicle binding increases nucleation around 5 million times! The authors have also been involved in another study indicating that different αSN oligomers interconvert enroute to the fibrils8; this conversion can also be included in the model, but the fit is very decent even without this step (Fig. 1). Where does this leave us? It is a significant advance that we can incorporate the role of lipids into fibrillation mechanisms at the mechanistic level. As is often the case, this is just the first step and much remains to be refined. Ultimately models like these are interesting only if they are physiologically relevant. That may be

questioned at this stage. Synaptic vesicles are not 100% anionic like the DMPS vesicles, but rather are complex lipid mixtures containing around 20% anionic lipids while the rest are zwitterionic or neutral9. Will complex mixtures have a similar effect? Moreover, the estimated synaptic lipid:αSN ratio in vivo is estimated to 400, well above the optimal ratio seen here. But perhaps higher lipid ratios could compensate for lower charge? Moreover, these experiments are conducted under conditions under which DMPS is in a state of flux between the ordered and disordered states: DMPS normally melts around 41 °C, but binding of αSN reduces this to ~31 °C, so that the lipid is actually in a rather ill-defined physical state; this is not necessarily unphysiological but adds to the complexity of the interpretation. A further mismatch is that all these exciting experiments were conducted at 0 mM NaCl; even 25–50 mM NaCl (well below physiological concentrations) strongly inhibit the effect of lipids. Reducing the salt concentration can also lead to αSN fibrils with very different structural and functional properties10, but although this illustrates the diversity of αSN conformations, the link to in vivo conditions seems rather doubtful.

Nevertheless, the article is a trailblazer and should inspire many interesting follow-up studies that in the end may lead to realistic models of what actually happens when αSN fibrillates in the cell. ■ Daniel Otzen is in the Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Center for Insoluble Protein Structures (inSPIN), Aarhus University, Aarhus, Denmark. e-mail: [email protected] References

1. Galvagnion, C. et al. Nat. Chem. Biol. 11, 229–234 (2015). 2. Spillantini, M.G. & Goedert, M. Ann. NY Acad. Sci. 920, 16–27 (2000). 3. Auluck, P.K., Caraveo, G. & Lindquist, S.L. Annu. Rev. Cell Dev. Biol. 26, 211–233 (2010). 4. Lorenzen, N. et al. J. Am. Chem. Soc. 136, 3859–3868 (2014). 5. Volles, M.J. & Lansbury, P.T. Biochemistry 42, 7871–7878 (2003). 6. Last, N.B., Rhoades, E. & Miranker, A.D. Proc. Natl. Acad. Sci. USA 108, 9460–9465 (2011). 7. Cohen, S.I., Vendruscolo, M., Dobson, C.M. & Knowles, T.J. in Amyloid Fibrils and Prefibrillar Aggregates: Molecular and Biological Properties (ed. Otzen, D.E.) 7–9 (Wiley-VCH, 2013). 8. Cremades, N. et al. Cell 149, 1048–1059 (2012). 9. Benfenati, F., Greengard, P., Brunner, J. & Bähler, M. J. Cell Biol. 108, 1851–1862 (1989). 10. Bousset, L. et al. Nat. Commun. 4, 2575 (2013).

Competing financial interests The author declares no competing financial interests.

NATURAL PRODUCTS

Untwisting the antibiotic’ome

Microbial natural products and the specific subset with antibiotic activity, ‘the antibiotic’ome’, consist of a dizzying array of structures and exert their effects by many known modes of action. In this issue, Cociancich et al. describe a unique natural product that—along with a compound identified in a recent publication by Baumann et al.—defines a new antibacterial chemical scaffold that acts on a rarely hit target, DNA gyrase subunit A.

Chad W Johnston & Nathan A Magarvey

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icrobial secondary metabolism has been a wellspring of chemical diversity and antibiotic lead compounds since the 1940s, providing thousands of bioactive molecules and the majority of clinically relevant antibacterial chemical scaffolds1. Two of the major biosynthetic classes present in this ‘antibiotic’ome’ are polyketides and nonribosomal peptides—complex small molecules produced by modular enzymatic assembly lines. Although these privileged compounds have traditionally been isolated from prolific producer families such as Bacilli, Actinobacteria and Fungi, widespread microbial genome sequencing is revealing similar biosynthetic potential from previously untapped sources. Given the current need for new antibacterial leads,

gaining access to new sources of microbial chemistry should be considered a high priority. In this issue of Nature Chemical Biology, Cociancich et al.2 describe the structure and biosynthesis of albicidin, a powerful antibiotic from the sugarcane microbe Xanthomonas albilineans. By heterologously expressing the biosynthetic genes required for the compound’s production, these researchers were able to unlock exotic chemistry from this similarly exotic organism, providing a potential lead for antibacterial drug development. Although Cociancich et al.2 are the first to elucidate the structure of albicidin, it has been known for decades as the phytotoxin responsible for leaf scald disease in sugarcane3. Genetic data obtained in 2001 first demonstrated that albicidin was

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a polyketide synthase–nonribosomal peptide synthetase (PKS-NRPS) product4, with the biosynthetic gene cluster including genes encoding both a trans-acting acyltransferase and a much rarer transacting NRPS5. Much like Pseudomonas syringae—another biosynthetically talented plant pathogen—X. albilineans appears to utilize carefully developed chemistry to gain a foothold in its choice environmental niche. Because it inhibits the function of DNA gyrase through interactions with the A subunit, albicidin’s actions are distinct from other natural-product DNA gyrase inhibitors that hit the B subunit. Before now, researchers had been unable to isolate sufficient quantities of albicidin for structure elucidation, likely because albicidin production is carefully controlled in the host 177

news & views N O O

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© 2015 Nature America, Inc. All rights reserved.

Figure 1 | New, exotic DNA gyrase inhibitors from the antibiotic’ome. After nearly 10,000 known polyketide and nonribosomal peptides have been surveyed, albicidin (top left) and the cystobactamids (bottom left) represent the first natural products to inhibit the A subunit of DNA gyrase—a clinically validated antibacterial target for synthetic quinolones.

and only nanomolar quantities are required for gyrase inhibition. To increase yields, a heterologous expression system was developed in the related species Xanthomonas axonopodis6, and this heterologous host served as a launch point for the current study. Along with optimization of the medium, this heterologous expression system improved the yield of albicidin to ~0.01 mg/L. Albicidin’s adherence to glass forced Cociancich et al.2 to use small plastic containers to culture the hundreds of liters of isotopically enriched medium necessary to isolate sufficient quantities of material for structure elucidation by mass spectrometry and NMR. As a reward for the authors’ heroic efforts, they discovered that albicidin possesses an unprecedented chemical scaffold (Fig. 1), including an unusual β-cyanoalanine and myriad aromatic δ-amino acids derived from chorismate and para-amino benzoate (pABA). Though nonribosomal peptides are considered to be reliably predictable through established substrate selection codes in the monomer-specifying adenylation domains7, albicidin structure predictions were typically made with notoriously low confidence5. This poor predictability is readily explained by albicidin’s unusual aromatic δ-amino

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acids and the structural requirements that are apparently mandated for their activation, including a shifted aspartic acid involved in coordinating amines during monomer adenylation7. A further mystery was the biosynthesis and activation of the cyanoalanine residue, which appears to take place through the essential trans-NRPS, composed of an adenylation and tethering domain, as well as a unique insertion that is likely involved in generating cyanoalanine from asparagine. By expressing and assaying the monomer preferences of each adenylation domain, Cociancich et al.2 were able to define which residues were likely formed by post-assembly-line modifications, leading to a robust model for albicidin biosynthesis. The only remaining structural ambiguity—a sole stereocenter found in the cyanoalanine residue—was definitively elucidated through total synthesis in a second publication8, firmly solving the structure of this challenging natural product as well as providing a means to generate structural diversity and access sufficient quantities of albicidin for biological assays. In a remarkable coincidence, Baumann et al. have simultaneously reported a similar series of natural products9— named the cystobactamids—from an unrelated Cystobacter sp. (Fig. 1). Exotic

natural products in their own right, the cystobactamids possess a rare nitro functionality as well as an unprecedented series of O-isopropoxylations. Both albicidin and the cystobactamids target DNA gyrase subunit A and in a sense are natural versions of the clinically important synthetic fluoroquinolone antibacterials. However, these newly discovered natural products possess superior inhibitory activity compared to the fluoroquinolone ciprofloxacin, and they are not affected by the qnrA1 quinolone-resistance mutation10. As the synthetic fluoroquinolone chemical space is well mapped, these new natural products offer a promising opportunity to revisit DNA gyrase inhibition as a means to combat challenging drug-resistant Gram-negative bacteria. Using the biosynthetic mechanisms explained by Cociancich et al.2, targeted naturalproducts programs can now be developed to tap this previously cryptic portion of the antibiotic’ome for further promising antibacterial leads. ■ Chad W. Johnston and Nathan A. Magarvey are in the Department of Biochemistry and Biomedical Sciences, the Department of Chemistry and Chemical Biology and the M.G. DeGroote Institute of Infectious Disease at McMaster University, Hamilton, Ontario, Canada. Nathan A. Magarvey is also a Canada Research Chair in Natural Products & Chemical Biology. e-mail: [email protected] References

Newman, D.J. & Cragg, G.M. J. Nat. Prod. 70, 461–477 (2007). Cociancich, S. et al. Nat. Chem. Biol. 11, 195–197 (2015). Birch, R.G. & Patil, S.S. Phytopathology 73, 1368–1374 (1983). Huang, G.Z., Zhang, L.H. & Birch, R.G. Microbiology 147, 631–642 (2001). 5. Piel, J. Nat. Prod. Rep. 27, 996–1047 (2010). 6. Vivien, E. et al. Antimicrob. Agents Chemother. 51, 1549–1552 (2007). 7. Stachelhaus, T., Mootz, H.D. & Marahiel, M.A. Chem. Biol. 6, 493–505 (1999). 8. Kretz, J. et al. Angew. Chem. Int. Edn. Engl. doi:10.1002/ anie.201409584 (12 December 2014). 9. Baumann, S. et al. Angew. Chem. Int. Edn. Engl. 53, 14605–14609 (2014). 10. Tran, J.H. & Jacoby, G.A. Proc. Natl. Acad. Sci. USA 99, 5638–5642 (2002). 1. 2. 3. 4.

Competing financial interests

The authors declare no competing financial interests.

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Natural products: untwisting the antibiotic'ome.

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