One Enzyme To Build Them All: Ring-Size Engineered Siderophores

Subscriber access provided by University of Chicago Library

One Enzyme to Build Them All – Ring Size Engineered Siderophores Inhibit Swarming Motility of Vibrio Sina Rütschlin, Sandra Gunesch, and Thomas Boettcher ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00084 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 14, 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 8 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

One Enzyme to Build Them All – Ring Size Engineered Siderophores Inhibit Swarming Motility of Vibrio Sina Rütschlin, Sandra Gunesch and Thomas Böttcher* #

Department of Chemistry, Konstanz Research School Chemical Biology, Zukunftskolleg, University of Konstanz, 78457 Konstanz, Germany. *Email: [email protected]

Supporting Information Placeholder ABSTRACT: Bacteria compete for ferric iron by producing siderophores and some microbes engage in piracy by scavenging siderophores of their competitors. The macrocyclic hydroxamate siderophore avaroferrin of Shewanella algae inhibits swarming of Vibrio alginolyticus by evading this piracy. Avaroferrin as well as related putrebactin and bisucaberin are produced by the IucC-like synthetases AvbD, C PubC, and BibC . Here, we established that they are capable of synthesizing not only their native product but also other siderophores. Exploiting this relaxed substrate specificity by synthetic precursors generated 15 different ring size engineered macrocycles ranging from 18- to 28-membered rings, indicating unprecedented biosynthetic flexibility of the enzymes. Two of the novel siderophores could be obtained in larger quantities by precursor-directed biosynthesis in S. algae. Both inhibited swarming motility of Vibrio and similar to avaroferrin the most active one exhibited a heterodimeric architecture. Our results demonstrate the impact of minor structural changes on biological activity, which may trigger the evolution of siderophore diversity.

Availability of the essential element iron is one of the most important factors limiting bacterial growth. In response to low solubility of ferric iron in the environment, bacteria have evolved to produce and secrete a wealth of small molecule chelators, called siderophores, with extremely high affinity 1 for ferric iron. The resulting siderophore-iron complexes are taken up by the cells via specific receptors. Since siderophores bind iron outside of the cells and the cell that profits from the iron uptake is not necessarily the same cell that 2, 3 produced the siderophore, they may present public goods. This makes siderophores susceptible to social cheating by bacteria that lack the costly pathways for siderophore biosynthesis but encode the corresponding uptake receptor – a 2, 4 phenomenon which is also known as siderophore piracy. The enormous competition for iron is consequently spurring the continuous evolution of new siderophores along with highly specific receptors. Not only are siderophores important for microbial interactions but they also play major roles as virulence factors of pathogenic bacteria in human 5, 6 and animal diseases. The hydroxamate siderophore desfer-

rioxamine B is even in medicinal use as drug for treating iron 7 overload in humans. While many siderophores are synthesized by nonribosomal peptide synthetases (NRPS), the elucidation of the biosynthesis of desferrioxamines in Streptomyces has led to the discovery of a novel class of NRPS8-10 independent siderophore (NIS) synthetases. Also dimeric macrocyclic siderophores are produced via some NIS synthetases, whereby these enzymes catalyze two separate steps: the initial dimerization of the substrates and the following 9, 11 macrocyclization reaction (Figure 1A).

Figure 1. A) Steps in the biosynthesis of dimeric macrocyclic siderophores catalyzed by some IucC-like type C NIS synthetases. Substrate molecules HS[X]A are first dimerized and finally macrocyclized to a symmetric or asymmetric siderophore [X+X’]. B) Structures of the three siderophores produced via AvbD in S. algae. C) Phylogenetic tree of IucC-like type C NIS synthetases with RhbC (type A NIS) of Sinorrhizobium meliloti serving as outgroup. Bootstrap values >70%

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

are indicated at the corresponding branches. The scale bar represents amino acid substitutions per site. We have previously discovered that the chimeric cyclic hydroxamate siderophore avaroferrin of Shewanella algae B516 inhibits swarming of Vibrio alginolyticus and concluded that 12 it does so by blocking siderophore piracy of Vibrio. In contrast, the homodimeric counterparts putrebactin and bisucaberin were considerably less active swarming inhibitors. We recently investigated the biosynthesis of these three related siderophores which are all produced by the same Shewanella algae B516 strain and differ only by the length of 13 their diamine building blocks (Figure 1B).

ed the synthetic and native precursors with AvbD, PubC and C BibC in ATP reaction buffer. Based on the amounts of macrocycles produced by each enzyme, we calculated the relative ratios (Supporting Information). All three enzymes were able to convert the artificial precursors alone or in combination with the native substrates to the corresponding homo- and heterodimeric macrocycles (Figure 2, Table S1).

We demonstrated that AvbD, a IucC-like type C NIS synthetase is responsible for the dimerization and macrocyclization of the monomeric precursors. AvbD showed a much higher affinity and catalytic efficiency for the larger cadaverine derived substrate HS[5]A leading to bisucaberin in comparison to the shorter putrescine derived HS[4]A, the precursor of homodimeric putrebactin. Yet, the cell’s substrate pool appeared to be adjusted to maximize production of the ad13 vantageous chimeric avaroferrin. Stimulated by these results, we were now interested to also investigate the specificity of the related dimerizationC macrocyclisation type C synthetases BibC and PubC, which were originally described to be the corresponding homologues for the biosynthesis of bisucaberin and putrebactin, 11, 14 respectively. To this aim, we first performed a detailed phylogenetic analysis of the type C NIS synthetases by maximum likelihood (Figure 1C, Figure S1). C

In the resulting tree, PubC, AvbD, and BibC homologues clustered in three separate groups, which together formed a sister clade to DesD and AlcC homologues, the biosynthesis enzymes of the trimeric siderophore desferoxamine and the dimeric hydroxylated siderophore alcaligin, respectively. C AvbD was hereby more closely related to BibC as compared to PubC homologues. The phylogenetic distance of PubC, C AvbD, and BibC raised the question if these differences would be also reflected in substrate discrimination of the enzymes towards the synthesis of their designated native siderophore products. We thus synthesized the two native biosynthetic precursors 11, 14, 15 HS[4]A and HS[5]A as described previously. We then C heterologously expressed PubC, AvbD, and BibC in E. coli and purified the enzymes to reconstitute the ATP-dependent enzyme reaction in vitro. Surprisingly, each of the enzymes was capable of producing all three siderophores, putrebactin, avaroferrin, and bisucaberin without major differences in substrate discrimination. We conclude, that in species using C PubC and BibC the available substrate pool of a cell dictates the siderophore ratios as we have demonstrated previously 13 for AvbD in S. algae B516. Accordingly, pure putrebactin producers lack the AvbA/BibA homologue of a lysine decarboxylase for cadaverine production and strains producing mainly bisucaberin supposedly have a bias towards generating the cadaverine-derived precursor. As rather substrate availability than specificity of the synthetase determines the production of the corresponding native siderophores by these IucC-like enzymes, we suspected that the enzymes might also accept other non-native substrates. To investigate this possibility, we synthesized precursor molecules HS[3]A to HS[6]A, and HS[8]A with different chain lengths in the diamine subunit (Supporting information). We then convert-

Figure 2. Macrocycle [X+X’] production by A) AvbD, B) C BibC and C) PubC given in relative amounts formed after 4 h with 4 µM enzyme (A) and (C) or 6 h and 8 µM enzyme (B) for conversion of combinations of substrates HS[X]A at 15°C C for AvbD and PubC and at 30°C for BibC . The number of


Page 2 of 8

Page 3 of 8 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 methylene-groups in the chain between the nitrogen atoms is given on the-x axis. All homodimeric products and heterodimeric combinatorial products could be detected with ring sizes ranging from 18to 38-membered macrocycles, which were identified based on high-resolution mass spectra (Table S1). Furthermore, all compounds were confirmed by fragmentation analysis with MS-MS (Figure S2). All three enzymes were relatively similar in accepting a broad range of non-natural substrates. PubC was slightly more capable of incorporating short-chain preC cursors than AvbD and BibC displayed the narrowest substrate range (Figure 2, Table S1). Surprisingly, macrocycle production was controlled on the level of substrate discrimination and not by the ring size of the product. While for example [3+8] was produced only in traces, [5+6] was formed in considerable quantities although both comprise a 23-membered ring. Also combinations of HS[3]A with other precursors yielded only small amounts of the heterodimeric products. While the native substrates HS[4]A and HS[5]A were generally preferred, non-natural HS[6]A was surprisingly well accepted by the enzymes and PubC yielded [6+6] and [5+6] even as the most abundant products. The heterodimeric [5+6] also was one of the major products of C AvbD and BibC . Our results thus indicate a general highly relaxed substrate selectivity for three homologous IucC-like synthetases, which may allow for rapid evolution of new siderophores. To test if the new macrocylic compounds also behaved as siderophores, we supplemented the samples with 10 mM aqueous solution of FeCl3 to observe mass shifts to 16 the respective iron-complexes (Table S2). For almost all artificial macrocycles produced, with the exception of [3+3] and [8+3], we could detect the corresponding 1:1 iron+ siderophore complexes [M+Fe] with their characteristic isotopic pattern, indicating formation of a tetradentate iron chelate. We also investigated chelation of other ions, includ3+ ing Ga , vanadate, and molybdate, which were all three found to form 1:1 complexes with the majority of macrocycles (Table S3). Having confirmed that most of the macrocyclic products were indeed siderophores, we speculated that some of these non-natural siderophores like native avaroferrin may also prevent Vibrio’s siderophore piracy and thereby inhibit swarming motility. However, as the reconstituted in vitro reactions did not yield sufficient amounts for further experiments, we explored the possibility of using the live cells of S. algae as bioreactors for precursor-directed siderophore biosynthesis. Cells were grown on carrageenan plates under iron starvation conditions and native putrescine (required for HS[4]A) synthesis was suppressed using an ornithine decar16 boxylase inhibitor. Feeding of different diamines was followed by solvent extraction and LC-MS analysis of the extracts. These resulted only for the artificial substrate hexane1,6-diamine in a considerable degree of product formation. Shewanella algae produced significant amounts of the nonnatural engineered siderophores [5+6], [4+6] and [6+6] next to the natural siderophores putrebactin [4+4], avaroferrin [4+5] and bisucaberin [5+5] (Figure 3, Figure S3, Table S4).

Figure 3. Production of siderophores by live cells of S. algae fed with hexane-1,6-diamine. A) Structures of newly produced siderophores. B) LC-MS trace of siderophore production of precursor-directed biosynthesis by Shewanella algae fed with 1,6-diaminohexane. EICs (Extracted Ion Chromato+ grams) are shown for putrebactin m/z = 373.20 [M+H] , + avaroferrin, m/z = 387.20 [M+H] , bisucaberin and [4+6] m/z + + = 401.22 [M+H] , [5+6] m/z = 415.23 [M+H] and [6+6] m/z = + 429.25 [M+H] . Two of the artificial siderophores [5+6] and [6+6] could be extracted in sufficient amounts and isolated. The products were purified to homogeneity by HPLC and the structures were characterized by 1D and 2D NMR spectroscopy and mass spectrometry (Figures S4-S15). Previously reported attempts of feeding artificial diamines of different chain lengths to another Shewanella strain did not result in prod17 uct formation, indicating that our strain S. algae B516 has an exceptionally relaxed substrate acceptance also in AvbB and AvbC, which are responsible for the HS[X]A precursor biosynthesis. We were now interested if the isolated new siderophores had effects on swarming inhibition of Vibrio. Previous work has demonstrated that swarming of Vibrio alginolyticus is dependent on availability of ferric iron and inhibition of swarming is mediated by iron-chelation. However, swarming inhibition did not correlate with the binding affinities for iron of the corresponding chelators. We reasoned that not the strength of iron chelation but rather the ability of V. alginolyticus to utilize the chelated iron determines the 12 activity of swarming inhibitors. The genome of V. alginolyticus encodes numerous siderophore importers for xenosiderophores, which it does not produce by itself and related Vibrio species are known to engage in siderophore piracy, the 18, 19 poaching of siderophores from competitors. We had thus postulated that in contrast to siderophores like desferrioxamine B that does not inhibit swarming, heterodimeric avaroferrin of S. algae thwarts piracy by Vibrio and cutting off Vibrio from its ferric iron resource halts its swarming 12 motility. We now reasoned, that non-natural siderophores like [5+6] and [6+6] may also escape siderophore piracy and thereby inhibit swarming of V. alginolyticus. To investigate this possibility, we performed a previously established swarming assay on carrageenan plates with the prolific swarming strain V. alginolyticus B522, which had been coisolated with avaroferrin producing S. algae B516 from the 12, 20 same specimen of marine macroalga. Along with the native controls avaroferrin and bisucaberin previously isolat-


ACS Chemical Biology 13

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

ed, the purified engineered siderophores were added on paper discs surrounding a swarming colony of V. alginolyticus and we monitored for the formation of a clear inhibition zone (Figure 4A). None of the compounds had antibacterial activities and also did not inhibit Escherichia coli and Bacillus subtilis up to 200 µM.

Figure 4. Swarming inhibition by ring-size engineered siderophores. A) Swarming assay with [5+6] and [6+6] in comparison to avaroferrin [4+5] and bisucaberin [5+5] at 50 nmol showing inhibition zones imaged at different time points. B) Time-resolved development of the clear zone as distance to the swarming front measured from the inoculated paper discs. C) Concentration dependent series of distance to front (zone diameter) after 8 hours. A first test revealed that the engineered siderophores [5+6] and [6+6] exhibited considerable swarming inhibition activity (Figure 4A). To test whether the engineered siderophores inhibited swarming as result of limiting bioavailablitiy of ferric iron, we added excess of 2-fold ferric iron onto the paper discs inoculated with the active siderophores (Figure S16). The inhibitory effect was reversed by addition of ferric iron confirming that [5+6] inhibited by the same mode of III action as previously established for avaroferrin. The pᴍ(Fe ) values of the new siderophores were comparable to those of putrebactin, avaroferrin, and bisucaberin, confirming again that not the binding affinities but the ability of V. alginolyticus to use the siderophore-bound iron are critical for swarming inhibition (Figure S17). To quantitate the effect of the engineered artificial siderophores in respect to avaroferrin, we monitored swarming inhibition in dependence of concentration and time. As shown in Figure 4A and B the size of clear swarming-inhibition zones [6+6] and [5+6] were considerably more persistent and larger compared to bisucaberin [5+5] whereby [5+6] was more active than [6+6]. Over the entire concentration range [5+6] was about as active as avaroferrin (Figure 4B). These results demonstrate that our ring-size engineered artificial siderophores are able to mimic the effect of native avaroferrin to inhibit swarming Vibrio by depriving the cells of ferric iron, which is required for swarming motility. It has been previously proposed that only those siderophores, which cannot be readily scavenged by V. alginolyticus are 12 active swarm inhibitors. It could thus be expected that swarming Vibrio is less likely capable of utilizing artificial ring size engineered siderophores, which we could now confirm. Interestingly, the [5+6] siderophore, resembling the

Page 4 of 8

heterodimeric architecture of avaroferrin was considerably more active than the homodimeric [6+6] siderophore akin to bisucaberin. It is thus conceivable, that the asymmetric structure of heterodimers is less well recognized by Vibrio. The fierce battle for scavenging ferric iron and thwarting siderophore piracy of their competitors has triggered the evolution of an enormous structural diversity of microbial 21, 22 A common theme in the evolution of sidersiderophores. ophores is the acquisition and mutation of modular genes 23-25 tailoring and modifying the siderophore’s structure. In other cases different independent gene clusters shuffled into one organism coordinate the production of novel sidero4, 26 phores as reported for rhodochelin or serratiochelins. Diversity can also be generated by multispecificity of enzymes in the biosynthesis incorporating multiple different 27 substrates. We recently demonstrated that this is the case for AvbD in S. algae whereby product ratios are dictated by 13 the available substrate pool. Our new results suggest that this concept is generally applicable to an entire subgroup of C IucC-like enzymes. PubC and BibC which had been previously investigated in context of their native products putre11, 14, 28 bactin and bisucaberin, do not drastically differ in their specificity from AvbD. All of them can be thus regarded to be broad-spectrum dimerization and macrocyclization enzymes. While previous attempts to generate artificial dimeric macrocyclic siderophores by precursor-directed biosynthesis 16 successfully led to unsaturated versions of putrebactin, we report here for the first time ring-size engineered variants ranging from 18- to 28-membered rings. This extraordinarily broad spectrum of macrocycles of different size and architecture that can be produced simply by feeding different precursors indicates an unprecedented potential of these enzymes in the evolution of structural diversity of siderophores. An important example of how already small changes to a siderophore can have major ecological or even pathophysiological consequences is illustrated by pathogenic strains of E. coli and Salmonella. These strains glycosylate and linearize the siderophore enterobactin, which allows them to evade sabo29 tage of iron scavenging by the human immune response. Accordingly, our results with artificial ring-size variants of dimeric hydroxamate siderophores demonstrate that minor changes to a siderophore core scaffold such variation of ringsize or heterodimeric versus homodimeric architecture can have significant effects on competing species, which here is illustrated by the inhibition of swarming Vibrio.

METHODS Additional methods can be found in the Supporting Information. Synthesis of N-hydroxy-N-succinyl-alkylenediamines. The corresponding HS[X]A substrates were produced from dihaloalkanes using Gabriel synthesis followed by substitution of the second halogen with tert-butyl N(benzyloxy)carbamate, partial de- and reprotection and coupling with succinic anhydride, according to a method 14 described previously. C

Overexpression and purification of AvbD, BibC and PubC. The proteins were recombinantly expressed with Nterminal Strep-tag in E. coli. Proteins were purified from


Page 5 of 8 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 StrepTrap™ HP affinity column (GE Healthcare Bio-Sciences AB) on an ÄKTA™ start protein purification system. Enzyme reactions. Substrates HS[X]A were incubated in 1:1 ratio (total 2 mM) with 4 mM ATP, 15 mM MgCl2, 25 mM Tris-HCl (pH 8.0) and 4 μM enzyme (AvbD and PubC) in a C final volume of 140 μL (or 70 µL for BibC ) for four hours at C 15°C. Reactions with BibC were carried out at 30 °C with 8 μM enzyme for 6 h. In aliquots of 40 µL, reactions were stopped by addition of 3 µL 10% trichloroacetic acid. LC-MS analysis of the reaction mixture was carried out as described in the Supporting Information. Precursor-directed biosynthesis and isolation of siderophores. S. algae B516 was grown on carrageenan NBE plates for 4 to 5 days at 30°C with 100 μM 2,2’-bipyridyl as iron chelator, 10 mM 1,4-diamino-2-butanone dihydrochloride as ornithine decarboxylase inhibitor and 10 mM 1,6diaminohexane. The agar was soaked overnight in ethyl acetate followed by isopropanol. The isopropanol extract was evaporated and the mixture was pre-fractionated on a SepPak SPE C18 cartridge (Waters) and subsequently purified by multiple preparative HPLC runs as detailed in the Supporting Information. Vibrio swarming assays. Standard swarming assays were performed on carrageenan NBE plates, prepared from 1.5 % (w/v) carrageenan for gel preparation in Nutrient Broth E. A 6 mm blank paper disc (BD BBL) was placed in the center of a plate surrounded by four further blank paper discs with 2 cm distance between the centers of each disc. The central disc was inoculated with 5 μL overnight culture of V. alginolyticus B522 in NBE medium and incubated at 30°C. After 12 h the surrounding discs were inoculated with 5 μL of a 10 mM DMSO stock of siderophores.

ASSOCIATED CONTENT Supporting Information

Syntheses, characterization of siderophores, mass spectra and bioassays. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author

* Email: [email protected] (T.B.) Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interests.

Funding Sources We gratefully acknowledge funding by the Emmy Noether program of the Deutsche Forschungsgemeinschaft (DFG), EU FP7 Marie Curie Zukunftskolleg Incoming Fellowship Program – University of Konstanz grant no. 291784, the Fonds der Chemischen Industrie (FCI), the Konstanz Research School Chemical Biology (KoRS-CB), and SFB969 (DFG). SR was supported by a KoRS-CB fellowship.

ACKNOWLEDGMENT We thank A. Marx and his group for their generous support and the Zukunftskolleg of the University of Konstanz. We thank N. Willassen and H. Hansen, UiT the Artic University of Norway for providing a genome sample of A. salmonicida LFI1238. We especially thank M. Prothiwa, H. Bußkamp, M. Mex and D. Hammler for their help with mass spectrometry.

REFERENCES (1) Miethke, M., and Marahiel, M. A. (2007) Siderophore-based iron acquisition and pathogen control, Microbiol Mol Biol Rev 71, 413-451. (2) Cordero, O. X., Ventouras, L. A., DeLong, E. F., and Polz, M. F. (2012) Public good dynamics drive evolution of iron acquisition strategies in natural bacterioplankton populations, Proc Natl Acad Sci U S A 109, 20059-20064. (3) West, S. A., Griffin, A. S., Gardner, A., and Diggle, S. P. (2006) Social evolution theory for microorganisms, Nat Rev Microbiol 4, 597607. (4) Seyedsayamdost, M. R., Cleto, S., Carr, G., Vlamakis, H., Joao Vieira, M., Kolter, R., and Clardy, J. (2012) Mixing and matching siderophore clusters: structure and biosynthesis of serratiochelins from Serratia sp. V4, J Am Chem Soc 134, 13550-13553. (5) Takase, H., Nitanai, H., Hoshino, K., and Otani, T. (2000) Impact of siderophore production on Pseudomonas aeruginosa infections in immunosuppressed mice, Infect Immun 68, 1834-1839. (6) Hirschmann, M., Grundmann, F., and Bode, H. B. (2017) Identification and occurrence of the hydroxamate siderophores aerobactin, putrebactin, avaroferrin and ochrobactin C as virulence factors from entomopathogenic bacteria, Environ Microbiol 19, 4080-4090. (7) Fisher, S. A., Brunskill, S. J., Doree, C., Gooding, S., Chowdhury, O., and Roberts, D. J. (2013) Desferrioxamine mesylate for managing transfusional iron overload in people with transfusion-dependent thalassaemia, Cochrane Database Syst Rev, CD004450. (8) Barona-Gomez, F., Wong, U., Giannakopulos, A. E., Derrick, P. J., and Challis, G. L. (2004) Identification of a cluster of genes that directs desferrioxamine biosynthesis in Streptomyces coelicolor M145, J Am Chem Soc 126, 16282-16283. (9) Kadi, N., Oves-Costales, D., Barona-Gomez, F., and Challis, G. L. (2007) A new family of ATP-dependent oligomerization-macrocyclization biocatalysts, Nat Chem Biol 3, 652-656. (10) Challis, G. L. (2005) A widely distributed bacterial pathway for siderophore biosynthesis independent of nonribosomal peptide synthetases, Chembiochem 6, 601-611. (11) Kadi, N., Arbache, S., Song, L., Oves-Costales, D., and Challis, G. L. (2008) Identification of a gene cluster that directs putrebactin biosynthesis in Shewanella species: PubC catalyzes cyclodimerization of N-hydroxy-N-succinylputrescine, J Am Chem Soc 130, 10458-10459. (12) Böttcher, T., and Clardy, J. (2014) A Chimeric Siderophore Halts Swarming Vibrio, Angew Chem Int Ed Engl 53, 3510-3513. (13) Rütschlin, S., Gunesch, S., and Böttcher, T. (2017) One Enzyme, Three Metabolites: Shewanella algae Controls Siderophore Production via the Cellular Substrate Pool, Cell Chem Biol 24, 598-604 e510. (14) Kadi, N., Song, L., and Challis, G. L. (2008) Bisucaberin biosynthesis: an adenylating domain of the BibC multi-enzyme catalyzes cyclodimerization of N-hydroxy-N-succinylcadaverine, Chem Commun (Camb) 0, 5119-5121. (15) Lifa, T., Tieu, W., Hocking, R. K., and Codd, R. (2015) Forward and reverse (retro) iron(III) or gallium(III) desferrioxamine E and ringexpanded analogues prepared using metal-templated synthesis from endohydroxamic acid monomers, Inorg Chem 54, 3573-3583. (16) Soe, C. Z., and Codd, R. (2014) Unsaturated macrocyclic dihydroxamic acid siderophores produced by Shewanella putrefaciens using precursor-directed biosynthesis, ACS Chem Biol 9, 945-956. (17) Soe, C. Z., Telfer, T. J., Levina, A., Lay, P. A., and Codd, R. (2016) Simultaneous biosynthesis of putrebactin, avaroferrin and bisucaberin by Shewanella putrefaciens and characterisation of complexes with iron(III), molybdenum(VI) or chromium(V), J Inorg Biochem 162, 207-215. (18) Kim, C. M., Park, Y. J., and Shin, S. H. (2007) A widespread deferoxamine-mediated iron-uptake system in Vibrio vulnificus, J Infect Dis 196, 1537-1545.



(19) Tanabe, T., Funahashi, T., Miyamoto, K., Tsujibo, H., and Yamamoto, S. (2011) Identification of Genes, desR and desA, Required for Utilization of Desferrioxamine B as a Xenosiderophore in Vibrio furnissii, Biol Pharm Bull 34, 570-574. (20) Böttcher, T., Elliott, H. L., and Clardy, J. (2016) Dynamics of Snake-like Swarming Behavior of Vibrio alginolyticus, Biophys J 110, 981-992. (21) Lee, W., van Baalen, M., and Jansen, V. A. (2012) An evolutionary mechanism for diversity in siderophore-producing bacteria, Ecol Lett 15, 119-125. (22) Hider, R. C., and Kong, X. (2010) Chemistry and biology of siderophores, Nat Prod Rep 27, 637-657. (23) Fischbach, M. A., Walsh, C. T., and Clardy, J. (2008) The evolution of gene collectives: How natural selection drives chemical innovation, Proc Natl Acad Sci U S A 105, 4601-4608. (24) Franke, J., Ishida, K., and Hertweck, C. (2014) Evolution of siderophore pathways in human pathogenic bacteria, J Am Chem Soc 136, 5599-5602. (25) Crosa, J. H., and Walsh, C. T. (2002) Genetics and assembly line enzymology of siderophore biosynthesis in bacteria, Microbiol Mol Biol Rev 66, 223-249.

(26) Bosello, M., Robbel, L., Linne, U., Xie, X., and Marahiel, M. A. (2011) Biosynthesis of the siderophore rhodochelin requires the coordinated expression of three independent gene clusters in Rhodococcus jostii RHA1, J Am Chem Soc 133, 4587-4595. (27) Wuest, W. M., Sattely, E. S., and Walsh, C. T. (2009) Three siderophores from one bacterial enzymatic assembly line, J Am Chem Soc 131, 5056-5057. (28) Oves-Costales, D., Kadi, N., and Challis, G. L. (2009) The longoverlooked enzymology of a nonribosomal peptide synthetaseindependent pathway for virulence-conferring siderophore biosynthesis, Chem Commun (Camb), 6530-6541. (29) Fischbach, M. A., Lin, H. N., Liu, D. R., and Walsh, C. T. (2006) How pathogenic bacteria evade mammalian sabotage in the battle for iron, Nat Chem Biol 2, 132-138.


Page 6 of 8

Page 7 of 8 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


7


TOC graphic 124x88mm (300 x 300 DPI)


Page 8 of 8

One Enzyme To Build Them All: Ring-Size Engineered Siderophores

Recommend Documents