ACS Chemical Biology - ACS Publications - American Chemical Society

Subscriber access provided by Nottingham Trent University

Article

Identification of novel #-pyrones from Conexibacter woesei serving as sulfate shuttles Franziska Wiker, Martin Konnerth, Irina Helmle, Andreas Kulik, Leonard Kaysser, Harald Gross, and Bertolt Gust ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00455 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 17, 2019

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 23 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

Identification of novel α-pyrones from Conexibacter woesei serving as sulfate shuttles Franziska Wiker†, Martin Konnerth‡, Irina Helmle†, Andreas Kulik◊, Leonard Kaysser†,§, Harald Gross†,§, Bertolt Gust*†,§ †Pharmaceutical

Institute, Department of Pharmaceutical Biology, University of Tübingen, Auf

der Morgenstelle 8, 72076 Tübingen, Germany ‡Organic

Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076 Tübingen,

Germany ◊Institute

of Microbiology and Infection Medicine, Auf der Morgenstelle 28, 72076 Tübingen,

Germany §German

Center for Infection Research (DZIF), partner site Tübingen, Germany

ABSTRACT: Pyrones comprise a structurally diverse class of compounds. Although they are widely spread in nature, their specific physiological functions remain unknown in most cases. We recently described that triketide pyrones mediate the sulfotransfer in caprazamycin biosynthesis. Herein, we report the identification of conexipyrones A-C, three previously unrecognized tetrasubstituted α-pyrones, from the soil actinobacterium Conexibacter woesei. Insights into their biosynthesis via a type III polyketide synthase were obtained by feeding studies using isotopeenriched

precursors.

In

vitro

assays

employing

the

genetically

associated

3'-

phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferase CwoeST revealed conexipyrones as the enzymes genuine sulfate acceptor substrates. Furthermore, conexipyrones were determined to function as sulfate shuttles in a two-enzyme assay, since their sulfated derivatives were accepted as donor molecules by the PAPS-independent arylsulfate sulfotransferase (ASST) Cpz4 to yield sulfated caprazamycin intermediates. Sulfation is a vital process in all living organisms. In bacteria, known biological functions of sulfation include cell-to-cell signaling, factors of virulence or modification of natural products. For cell-to-cell signaling, not only communication between members of the same species, but also with other bacteria and with eukaryotes could be observed.1 For example, signaling between the nodulating bacterium Sinorhizobium meliloti and its symbiotic host organism, the alfalfa plant, is mediated by a sulfated metabolite, the Nod factor.2 From studies on the 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 23

biosynthesis of sulfolipid-1 in Mycobacterium tuberculosis, sulfation was shown to be essential for the function of this molecule as a major virulence factor.3 Though natural product modification by sulfation is relatively uncommon, several sulfotransfer reactions in the bacterial secondary metabolism have been discovered over the last decades. This includes the sulfation of glycopeptide A47934, resulting in decreased induction of resistance in the environmental model organism S. coelicolor.4 Furthermore, sulfation takes place in the biosynthesis of liponucleoside antibiotics liposidomycins5, A-902896 and caprazamycins (CPZs).7 However, an impact on the antimicrobial activity of these molecules due to the sulfation has not been described yet. Sulfotransferases (STs) catalyze the transfer of a sulfate group from a donor molecule to an acceptor substrate. Regarding their specificity toward the donor, sulfotransferases are classified into

3´-phosphoadenosine-5´-phosphosulfate

(PAPS)-dependent

or

PAPS-independent

enzymes. Compared to the widely studied eukaryotic PAPS-dependent STs, only a small number of prokaryotic PAPS-dependent STs have been described in literature. The even less prevalent PAPS-independent arylsulfate sulfotransferases (ASSTs) comprise a virtually uncharacterized family of STs employing two aryl substrates, one as a sulfate donor and one as an acceptor. ASSTs have so far only been reported from microorganisms and fungi and were initially discovered from commensal intestinal bacteria.8 Recently, a novel two-step sulfation mechanism involving both kinds of sulfotransferases was discovered during the biosynthesis of sulfated CPZ derivatives.9 Herein, the type III polyketide synthase (PKS) Cpz6 is involved in the biosynthesis of a family of new triketide pyrones, which were named presulficidins (Figure 1A (4)). In the following, presulficidins are sulfated by the PAPS-dependent ST Cpz8 to yield phenolic substrate esters, the sulficidins. In a final step, the PAPS-independent ASST Cpz4 transfers the sulfate group from the sulficidins onto hydroxyacylcaprazols (5), caprazamycin aglycones or caprazamycins to generate sulfated analogues (6). This discovery describes an intriguing new function for pyrones as sulfate shuttles in secondary metabolism. Especially in bacteria, specific physiological functions could be characterized only for a few pyrone derivatives, including cell-to-cell signaling and defense against competitors and predators. Germicidins were the first known autoregulative inhibitors of spore germination in the genus Streptomyces.10-12 Photopyrones, produced by an entomopathogenic bacterium species, Photorhabdus luminescens, represent another class of extracellular signaling molecules in bacterial cell-cell communication.13 Hence, we were interested, if the discovered role of pyrones as sulfate shuttles is a one-case scenario or exists in other organisms as well. To this end, we conducted a genome mining 2 ACS Paragon Plus Environment

Page 3 of 23 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


approach identifying several bacterial strains with the genetic ability to employ a two-step sulfation mechanism. In vivo and in vitro analysis of the discovered type III PKS and PAPSdependent ST genes from Conexibacter woesei, homologs to Cpz6 and Cpz8, respectively, lead to the identification of the herein described novel conexipyrones. Conexipyrones were further confirmed to function as sulfate shuttles in a sulfation mechanism analogous to that in caprazamycin biosynthesis. RESULTS AND DISCUSSION Genome mining. The genes cpz4 (ASST), cpz6 (type III PKS) and cpz8 (PAPS-dependent ST), essential for a two-step sulfation process, were used for an individual BLAST homology search.14 Strains that showed hits for all three genes were subjected to a combined BLAST search using MultiGeneBlast. Amongst others, we identified a two-gene cluster in Conexibacter woesei DSM 14684.15, 16 Here, a PAPS-dependent ST and type III PKS homolog are located directly adjacent to each other, while a homolog of the ASST is situated on a different locus 196 kb upstream of the former genes (Figure 1B). In the presented work, we focused on the investigation of a possible sulfation mechanism in Conexibacter woesei. Hence, the respective function for each of the found homologous genes was assigned according to a BLAST homology search (Figure 1B). Since Cwoe4977, which will further be referred to as CwoePKS, exhibits a 31% identity to Cpz6, one could speculate that the encoded type III PKS may produce compounds related to the presulficidins comprising a similar function. Conexipyrones are products of CwoePKS. For identification of putative new products generated by CwoePKS, we introduced the plasmid pFW03, containing the respective gene under the control of the strong constitutive promoter PermE*, and pUWL-apra-oriT as a control into strain S. coelicolor M145/Δsco7221, containing a knockout of the type III PKS, responsible for germicidin biosynthesis. Analysis of crude extracts from S. coelicolor M145/Δsco7221/pFW03 via HPLC revealed the production of one major compound (A) and five minor analogues (B-F), compared to that of the mutant strain containing the empty vector (Figure 2). Therefore, we purified all six compounds via preparative and subsequent semi-preparative HPLC. HR-MS analysis revealed C11H17O3 (measured m/z 197.1170 [M+H]+, calc. for C11H17O3, 197.1172, ∆-1.0 ppm, Supplementary Figure 1), C12H18NaO3 (measured m/z 233.1151 [M+Na]+, calc. for C12H18NaO3, 233.1148, ∆+1.3 ppm, Supplementary Figure 8), C13H20NaO3 (measured m/z 247.1306 [M+Na]+, calc. for C13H20NaO3 247.1305, ∆+0.3 ppm, Supplementary Figure 14), 3 ACS Paragon Plus Environment


Page 4 of 23

C10H14NaO3 (measured m/z 205.0837 [M+Na]+, calc. for C10H14NaO3, 205.0835, ∆+0.8 ppm, Supplementary Figure 21), C11H14NaO3 (measured m/z 217.0839 [M+Na]+, calc. for C11H14NaO3, 217.0835, ∆+1.8 ppm, Supplementary Figure 22) and C12H16NaO3 (measured m/z 231.0994 [M+Na]+, calc. for C12H16NaO3,, 231.0992, ∆+0.8 ppm, Supplementary Figure 23) as the molecular formulas of compounds A-F, respectively. Since the masses and molecular formulas of compounds A and D matched those of already described germicidin derivatives11, we suggested a similar pyrone structure and thus named the substances as conexipyrones A-F. The following in depth structure elucidation of conexipyrones was mainly based on 1D and 2D NMR experiments. However, purity or quantity of conexipyrones D, E and F were insufficient to obtain conclusive results. Therefore, merely structure elucidation of conexipyrones A, B and C was feasible (1-3) (Figure 1A, Supplementary Table 1, Supplementary Figures 2-7, 9-13 and 15-19). IR absorptions and UV maxima indicated the presence of a typical α-pyrone moiety as the core structure for all three compounds. This was supported by the lowfield 13C resonances observed for C-1, C-3 and C-5 in the 13C NMR spectrum (δc 159.5–168.5). The α-pyrone ring system was readily constructed on the basis of 1H, 1H-13C heteronuclear single quantum coherence (HSQC) data and 1H-13C heteronuclear multiple bond (HMBC) correlations. Furthermore, 1H-13C-HMBC cross peaks from 2-CH3 to C-1, C-2 and C-3 and from 4-CH3 to C-3, C4- and C-5 determined the position of two methyl groups at the pyrone ring. The side chain of the different compounds was confidently assigned on the basis of 1H-1H-COSY and 1H-13C-HMBC

NMR correlations as isobutane in case of 1, isopentyl in case of 2 and 3-

methylpentyl in case of 3. At last, 1H-13C-HMBC cross peaks from H2-6 to C-5 connected the respective aliphatic chains to the ring chromophore. Therefore, conexipyrones A-C were identified as previously undescribed tetra-substituted α-pyrones. High resolution LC-ESI/MS analysis of C. woesei crude extract revealed that also the wild-type strain produces conexipyrone A (Supplementary Figure 20). Based on the obtained molecular formulas for conexipyrones D, E and F, it is conceivable that these compounds represent analogues of conexipyrone A (1) and B (2) with a shorter and unsaturated side chains, respectively, (Supplementary Figures 21-23) similar to nocapyrones E-G17 or salinipyrone A.18 Feeding studies and proposed conexipyrone biosynthesis. Inspection of the structures of conexipyrones A-C (1-3) suggested that they might be assembled from different CoA- or ACPactivated iso-acyl starter units originated from branched-chain fatty acid metabolism, followed by elongation with two methylmalonyl units to result in the formation of the final triketide 4 ACS Paragon Plus Environment

Page 5 of 23 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


pyrones. To support this assumption, we conducted feeding experiments with isotope-enriched precursors. Labeled substrates were added at a final concentration of 5 mM to cultures of S. coelicolor M145/Δsco7221/pFW03 after 24 h of cultivation. After feeding of [1-13C]-labeled propionate, MS analysis revealed the incorporation of two carbon atoms from this precursor into conexipyrone A (m/z 198.9 [M+H+2]+), conexipyrone B (m/z 212.9 [M+H+2]+) and conexipyrone C (m/z 226.9 [M+H+2]+) (Figure 3), supporting the hypothesis of pyrone ring formation from two methylmalonyl extender units. Subsequent feeding of [1,2-13C2]-labeled acetate resulted in an increase of the peaks at m/z 212.9 ([M+H+2]+) for conexipyrone B and m/z 226.9 ([M+H+2]+) for conexipyrone C. Supplementation of

L-leucine-1,2-13C2,

L-valine-13C5

and

L-isoleucine-13C6

lead to

incorporation of one carbon in conexipyrone A (m/z 197.9 [M+H+1]+), four carbons in conexipyrone B (m/z 214.9 [M+H+4]+) and five carbons in conexipyrone C (m/z 229.9 [M+H+5]+) from the respective precursors. These results indicated that the varying side chains of the pyrones are generated from starter units originating from branched-chain fatty acid metabolism. To further confirm the proposed biosynthesis, we isolated isotope-enriched conexipyrone A, as the main product, from cultures of S. coelicolor M145/Δsco7221/pFW03 supplemented with either [1-13C]propionate or [1,2-13C2]L-leucine. 13C-NMR spectroscopy revealed labeling of the pyrone ring at positions C-1 and C-3 with [1-13C]propionate, due to the significant enrichment of the corresponding carbon signals. Moreover, feeding of [1,2-13C2]L-leucine led to an enhancement of the

13C

NMR signal of C-5 (Supplementary Table 2 and Supplementary

Figures 24-25). Based on these results, we propose the following biosynthetic pathway for conexipyrones (Figure 4). Originating from amino acids L-leucine, L-valine and L-isoleucine, different acylCoA or -ACP starter substrates are formed. It is well known that isovaleryl-CoA, for instance, is generally derived from leucine degradation involving a transamination step and a subsequent oxidative decarboxylation by the branched-chain α-keto-acid dehydrogenase complex (Bkd), which can be found in Streptomyces19, as well as in Conexibacter woesei. Formation of isobutyryl-CoA and 2-methylbutyryl-CoA is analogously achieved via degradation of valine and isoleucine.20 Subsequently, isovaleryl-CoA in case of conexipyrone A (1), isocaproyl-CoA in case of conexipyrone B (2) and 4-methylcaproyl-CoA in case of conexipyrone C (3) is elongated with two extender units of methylmalonyl-CoA via Claisen condensation to yield a linear polyketide intermediate. This is rather rare, since in the biosynthesis of most of the type III PKS derived metabolites one malonyl-CoA unit and one methylmalonyl- or ethylmalonyl5 ACS Paragon Plus Environment


Page 6 of 23

CoA unit are employed as extender units, usually yielding tri-substituted products, such as presulficidins9 or germicidins11. Afterwards, cyclization of the intermediate is achieved through a lactonization, to afford the α-pyrone moiety. Structurally closest related to the conexipyrones are, e.g., the nocapyrones, especially nocapyrone R17, salinipyrone A21 and saccharomonopyrone B.22 Interestingly, all three mentioned examples for tetra-substituted α-pyrones are produced by marine Actinomycetes strains. In contrast, tri-substituted derivatives have so far only been described from soil bacteria, making conexipyrones the first tetra-substituted α-pyrones from terrestrial Actinomycetales. CwoeST is a PAPS-dependent sulfotransferase. Genome mining results revealed that the gene encoding Cwoe4978, which is further referred to as cwoeST, is located directly adjacent to cwoePKS, and shows an identity of 44% on amino acid level to the PAPS-dependent ST Cpz89. Interestingly, like Cpz8, CwoeST apparently lacks the conserved 5’-phosphosulfatebinding loop, which is commonly regarded a quintessential feature of all known PAPSdependent STs.23 To confirm, if CwoeST can indeed act as a sulfotransferase, we cloned and subsequently expressed the respective gene in E. coli, which yielded a soluble protein that was purified afterwards (Supplementary Figure 26). Purified protein was added to a reaction mixture with PAPS as sulfate donor and p-nitrophenol (pNP) as an acceptor. HPLC analysis detected the formation of p-nitrophenol sulfate (pNS) in comparison with the assay missing the enzyme and was verified via LC-ESI/MS2 (Supplementary Figure 27). Similar to Cpz8, different non-genuine sulfate acceptors were readily accepted by CwoeST indicating a similar substrate specificity of both sulfotransferases (Supplementary Table 3). However, in assays employing 4-metylumbelliferyl sulfate (MUS) or pNS as sulfate donors, no product formation could be observed (Supplementary Figure 28). Our biochemical data therefore strongly support that CwoeST functions as a PAPS-dependent sulfotransferase. To explore, whether conexipyrones are the genuine sulfate acceptors of CwoeST, we incubated the purified compounds in a reaction mixture together with PAPS and the enzyme. Subsequent LC-ESI/MS and MS2 analyses proved that conexipyrone A, B and C were readily sulfated by CwoeST, identifying the obtained products as sulfated conexipyrones (sulfCP A with m/z 275 [M-H]- at tR = 6.3 min (Figure 5), sulfCP B with m/z 289 [M-H]- at tR. 7.5 min and sulfCP C with m/z 303 [M-H]- at tR. 8.2 min) (Supplementary Figure 29). High resolution LC-ESI/MS analysis of C. woesei culture extracts further corroborated this hypothesis, as production of sulfated conexipyrone A was observed in the wild-type strain (Supplementary Figure 30). 6 ACS Paragon Plus Environment

Page 7 of 23 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


Conexipyrones serve as sulfate shuttles. As the conexipyrones bare a high structural similarity to the presulficidins, one could assume that their sulfated derivatives can also function as sulfate donors for Cpz4, the ASST from caprazamycin biosynthesis, to yield sulfated caprazamycin derivatives. To prove this hypothesis, we performed an in vitro two-enzyme assay containing purified CwoeST and Cpz4 together with PAPS, hydroxyacylcaprazol E (5) and conexipyrone A, B or C (1-3) (Figure 1A, Figure 6A). Product formation (6) was observed by LC-ESI/MS2 analysis. In the UV-chromatogram of the assay containing all components, a new peak with a retention time of 10.3 min was detected (Figure 6A). The mass spectrometric data of the new compound (6) revealed a parent ion at m/z 880.5 [M-H]-, corresponding to the addition of a sulfate (+80 Da) to the hydroxyacylcaprazol E (m/z 800.5 [M-H]-) (5). The sulfation was assigned to position 2’ of the hydroxyacylcaprazol-uridyl-moiety by comparative LC-HRMS, MS2 and LC-HRisCID-MRM (MS3) fragmentation analysis of (5) and (6) (Supplementary Figures 31-33). Additionally, LC-HRMS/MS analysis of sulfated hydroxyacylcaprazol A (7) and non-sulfated hydroxyacylcaprazol A and sulfated caprazamycin A aglycon (8) were taken into account and corroborated position 2’ as sulfation-site (Supplementary Figures 34-35). Sulfation position 2’ was confirmed by comprehensive 1Dand 2D-NMR analysis of (7) (Supplementary Table 4 and Supplementary Figures 36-41). Interestingly, only trace amounts of sulfated conexipyrone A could be observed by LC-ESI/MS when 1, CwoeST, Cpz4 and PAPS were existent in the reaction mixture (Supplementary Figure 42B), whereas exclusion of Cpz4 lead to accumulation of the sulfated derivative. This demonstrates that the sulfated compound is readily accepted by Cpz4 as a donor substrate. Accordingly, product formation of 6 was dependent on all components. In assays missing either CwoeST, Cpz4, PAPS or (1), production of sulfated hydroxyacylcaprazol E could not be detected (Figure 6A and Supplementary Figure 42A). The same results were observed when 1 was replaced with conexipyrone B (2) or C (3). These findings indicate that conexipyrones A-C can indeed act as sulfate shuttles in a two-step sulfation mechanism. To test whether CwoeST is able to accept other substrates than the genuine conexipyrones as a sulfate acceptor, an assay with presulficidin B (4), a described sulfate acceptor substrate for Cpz89, was additionally performed. LC-ESI/MS analysis showed that presulficidin B was also readily accepted by CwoeST as an acceptor substrate, since product formation of 6 was observed (Figure 6A and Supplementary Figure 42B). Since a cpz4 homologous gene, coding for a putative ASST, was also found in the genome of C. woesei, one could speculate that a process similar to that in caprazamycin biosynthesis is employed in C. woesei, as well. However, Cpz4 shows substrate specificities towards 7 ACS Paragon Plus Environment


Page 8 of 23

compounds that contain a (+)-caprazol core skeleton linked to a fatty acyl side chain. Genes responsible for the formation of such a skeleton are not present in C. woesei making it unlikely that a similar reaction is catalyzed by the Cpz4 homologue CwoeASST. Furthermore, the genes in the neighborhood up- and downstream of the putative ASST mostly exhibit functions attributed to primary metabolism. Therefore, a genuine acceptor substrate of the ASST from C. woesei remains unknown at present. Conclusions. Through a genome mining approach, we found several strains with the genetic capacity to employ a two-step sulfation mechanism. Heterologous expression of the type III PKS found in C. woesei lead to the identification of previously unknown tetra-substituted conexipyrone A-C. It was confirmed by in vitro experiments that they are sulfated by the sulfotransferase CwoeST in a PAPS-dependent manner, suggesting that they are the genuine sulfate acceptor substrates for the sulfotransferase. Two-enzyme assays containing CwoeST, Cpz4, PAPS, hydroxyacylcaprazol E (5) and conexipyrone A, B or C (1-3) showed that sulfated conexipyrone derivatives can also function as sulfate shuttles, as they were readily accepted by Cpz4 as a donor substrate to yield sulfated hydroxyacylcaprazol E (6). Combining highresolution mass spectrometry fragmentation analysis with 1D and 2D NMR spectroscopy, sulfation by Cpz4 was assigned for the first time to position 2’ of the hydroxyacylcaprazoluridyl-moiety and not to the aminoribose moiety as proposed in earlier studies.5,

7, 9

These

findings additionally expand the range of genuine donor substrates for ASSTs. So far, sulficidins were the only known genuine donor substrates for this class of sulfotransferases. Since sulfated conexipyrone A (1) is also found in extracts of the native producer C. woesei, a two-step sulfation process employed by the wild-type strain can be proposed. In this context, the identification of a putative substrate for the final sulfation poses an intriguing topic. For the identification of such a metabolite, further experiments will have to be conducted in the future. Furthermore, one might have to consider that conexipyrones could exhibit other biological functions apart from their proposed role as sulfate shuttles.

METHODS Bacterial strains and general methods. S. coelicolor M145/Δsco7221 11 and its mutant strains were cultivated on MS agar (2% soy flour, 2% mannitol, 2% agar; Carl Roth) or in TSB medium (BD Biosciences). LB medium (Carl Roth) was used for E. coli growth. In all cases, the appropriate antibiotics were added to the media. DNA isolation and manipulation were carried out applying standard methods for E. coli and Streptomycetes. 8 ACS Paragon Plus Environment

Page 9 of 23 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


Construction of CwoePKS expression plasmid. cwoePKS was amplified from genomic DNA of C. woesei with primers cwoePKS_HindIII_F and cwoePKS_SpeI_R (Supplementary Table 5). The 1.12 kb PCR product was cloned into the expression vector pUWL-apra-oriT taking advantage of the HindIII/SpeI restriction sides. The resulting plasmid, pFW03 was confirmed via sequencing and transferred into the non-methylating E. coli strain ET12456/pUZ8002. Intergeneric conjugation with S. coelicolor M145/Δsco7221 finally yielded S. coelicolor M145/Δsco7221/pFW03 and S. coelicolor M145/Δsco7221/pUWL-apra-oriT (empty vector as control). Production of conexipyrones. 50 mL of TSB media were inoculated with spores of S. coelicolor M145/Δsco7221/pFW03. The culture was incubated at 30°C at 200 rpm. After two days, 5 mL of the culture were added to 70 mL of the production medium, containing 1% soytone, 1% soluble starch and 2% D-maltose. The culture was grown at 30°C at 220 rpm for 7 days. Purification of conexipyrones. Conexipyrones were purified from 2 L supernatant of a S. coelicolor M145/Δsco7221/pFW03 culture. The supernatant was adjusted to pH 5.0 with 1 M HCl and extracted twice with an equal volume of ethyl acetate followed by evaporation to dryness. The residual substance was dissolved in 10 ml methanol. For HPLC (Agilent 1200 series) analysis a Reprosil-Pur Basic C18 (Dr. Maisch, 5 μm, 250 × 2 mm) column was used at a flow rate of 0.2 mL/min with a linear gradient as follows (A is 0.1% formic acid in water and B is 0.06% formic acid in acetonitrile): 0 min, 25% B; 35 min, 100% B; 35–40 min, 100% B; 40–41 min, 25% B; 41–50 min, 25% B. Detection was carried out at 290 nm. For further purification, the extract was separated via reverse-phase preparative HPLC using a Unisol C18(2) column (Agela Technologies, 250 x 21.2 mm, 5μ) connected to a Waters liquid chromatography system (Waters 2545 Quaternary Gradient Module, 2489 UV/Visible Detector, PrepInjector). The linear gradient was as follows: 50% B to 60 % B in 15 min at a flow rate of 10 mL/min (A is 0.1% formic acid in water and B is 0.06% formic acid in acetonitrile). Detection was carried out at 290 nm. Eluates containing the desired compounds were evaporated to dryness and submitted to another purification step. Pure conexipyrone A was obtained by semi-preparative normal-phase HPLC using a Phenomenex Luna silica column (5 µm, 250 x 4.6 mm) with a linear gradient as follows (A is 1-octanol and B is ethyl acetate): 0 min, 33% B; 15 min, 73% B; 16 min, 100% B; 16–20 min, 100% B; 21 min, 33% B; 21–23 min, 33% B.

9 ACS Paragon Plus Environment


Page 10 of 23

Pure conexipyrone B was obtained by semi-preparative reversed-phase HPLC using a Phenomenex Kinetex PFP column (5 µm, 250 x 4.6 mm) with a linear gradient as follows (A is 0.1% formic acid in water and B is 0.06% formic acid in acetonitrile): 0 min, 30% B; 12 min, 34% B; 13 min, 100% B; 13–18 min, 100% B; 19 min, 30% B; 19–23 min, 30% B. Pure conexipyrone C was obtained by semi-preparative reversed-phase HPLC using a Phenomenex Kinetex PFP column (5 µm, 250 x 4.6 mm) with a linear gradient as follows (A is 0.1% formic acid in water and B is 0.06% formic acid in acetonitrile): 0 min, 40% B; 6 min, 42% B; 7 min, 100% B; 8–13 min, 100% B; 14 min, 30% B; 14–18 min, 30% B. Detection was carried out at 290 nm. Structure elucidation of conexipyrones. High-resolution LC-ESI/MS analysis was carried out on a Bruker Daltonics MaXis 4G connected to a Thermo Scientific Ultimate 3000 system using a reversed-phase Reprosil 100 C18 column (3µm, 100 x 3 mm) at a flow rate of 0.5 mL/min. For separation of C. woesei culture extracts, a linear gradient from 10 to 100 % of solvent B for 35 min was used (solvent A: 0.1 % formic acid in water; solvent B: 0.06 % formic acid in methanol). NMR spectra were recorded on a BRUKER Avance III HD 400 MHz NanoBay NMR spectrometer (1H: 400 MHz; 13C: 101 MHz) at 293 K. Chemical shifts were determined relative to the solvent as internal standard (CD3OD, δH/δC 3.31/49.0). All 13C NMR spectra of 13C

enriched versions of conexipyrone A were recorded by using inverse-gated decoupling

(zgig pulse program (Bruker); d1=2 s). UV spectra were recorded in methanol on a Lambda 25 UV-Visible Spectrophotometer (Perkin Elmer). Infrared spectra were obtained on a Jasco FT/IR-4100 spectrometer. Optical rotation values were determined with a Jasco P-2000 Polarimeter (d = 1 cm). Conexipyrone A (1) 3-Hydroxy-5-isobutyl-2,4-dimethyl-1H-pyran-1-one; amorphous white solid; UV (MeOH) λmax (log ε) 205 (4.74), 291 (3.96) nm; IR νmax 3456, 2959, 1659, 1560 cm-1, 1H and 13C NMR data (Supplementary Figures 2-7); HRESIMS measured m/z 197.1170 [M+H]+, calc. for C11H17O3, 197.1172, ∆-1.0 ppm (Supplemental Figure 1). Conexipyrone B (2) 3-Hydroxy-5-isopentyl-2,4-dimethyl-1H-pyran-1-one; amorphous white solid; UV (MeOH) λmax (log ε) 204 (4.61), 290 (3.91) nm; IR νmax 3640, 2954, 1651, 1556 cm-1, 1H and 13C NMR data (Supplementary Figures 9-13); HRESIMS measured m/z 233.1151 [M+Na]+, calc. for C12H18NaO3, 233.1148, ∆+1.3 (Supplemenatry Figure 8). Conexipyrone C (3) 10 ACS Paragon Plus Environment

Page 11 of 23 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


3-Hydroxy-2,4-dimethyl-5-(3-methylpentyl)-1H-pyran-1-one; amorphous white solid; [ ∝ ]23 𝐷 +16° (c 0.25, LC-MS grade MeOH); UV (MeOH) λmax (log ε) 205 (4.45), 290 (3.69) nm; IR νmax 3656, 2960, 1653, 1552 cm-1, 1H and 13C NMR data (Supplementary Figures 1519); HRESIMS measured m/z 247.1306 [M+Na]+, calc. for C13H20NaO3 247.1305, ∆+0.3 ppm (Supplementary Figure 14). Determination of the sulfation site. Analytes were subjected to HR-isCID-MRM (MS3) in negative ion mode. Source parameters of a Bruker Daltonics MaXis 4G mass spectrometer were set as follows: endplate offset: 500V, capillary: 3000V, nebulizer: 2.0 bar, dry gas: 8.0 L/min, dry temp: 200°C, isCID energy: 120 eV, collision energy: 30.0 eV, xAcq.: 1.0; width 4.0, MRM: m/z = 331.0603 and m/z = 480.1292. Analytes were subjected to chromatographic separation on a Thermo Scientific/DIONEX UltiMate 3000 UHPLC system with diode array detector (DAD) prior to MS analysis. Column: Macherey-Nagel Nucleoshell EC RP-C18 (2.7 µm, 150 x 2 mm). Flow rate: 0.3 mL/min. Eluent: system A: H2O + 0.1% formic acid, system B: methanol + 0.06% formic acid. Gradient: 0 min (10% B), 20 min (100% B), 25 min (100% B), 26 min (10% B), 30 min (10% B). NMR-spectra for determination of the sulfation site were recorded on a Bruker Avance III HDX 700 (1H: 700 MHz, 13C: 176.09 MHz). Samples were solved in d6-DMSO. Feeding studies with isotope-labeled precursors. For feeding studies, cultures of S. coelicolor M145/Δsco7221/pFW03 were cultivated as described above. Aqueous solutions of the respective isotope-enriched precursors, i.e. [1-13C]propionate, [1,2-13C2]L-leucine, [13C5]Lvaline- and [13C6]L-isoleucine, were added to the main cultures at a final concentration of 5 mM after 24 h of incubation time. Culture extracts were monitored via LC-ESI/MS at a LC/MSD Ultra Trap System XCT 6330 (Agilent Technology) using a Nucleosil 100 C18 column (3 μm, 100 × 2 mm fitted with a pre-column 10 x 2 mm) at a flow rate of 0.4 mL/min with a linear gradient from 0 to 100 % of solvent B for 20 min (A is 0.1 % formic acid in water; B is 0.06 % formic acid in acetonitrile). Additionally, purified labeled conexipyrone A was additionally analyzed via NMR. The enrichment ratio of individual carbon atoms of the labeled product was calculated via comparison of

13C

signal intensities between

13C-labeled

and

unlabeled conexipyrone A. The values were normalized by using the enrichment ratio of C-7, as no

13C

incorporation was expected at this position. The enhancement factor (EHF) was

calculated by dividing the normalized signal intensity of

13C-labeled

conexipyrone A by the

normalized signal intensity of the unlabeled compound.



Page 12 of 23

Construction of CwoeST expression plasmid. cwoeST was ordered as a gBlock with adjacent restriction sites for EcoRI and XhoI (Integrated DNA Technologies Inc., Supplementary Table 4). The 660 bp dsDNA fragment was cloned into pHis8 taking advantage of the EcoRI/XhoI restriction sides. The resulting plasmid, pFW05, was confirmed via sequencing and transferred into E. coli Rosetta2 (DE3) pLysS. Isolation and purification of CwoeST. E. coli Rosetta2 (DE3) pLysS harboring pFW05 was cultivated in 1 L TB broth supplemented with 25 μg/mL chloramphenicol and 50 μg/mL kanamycin at 37°C at 220 rpm until an OD600 of 0.6. Afterwards, the temperature was shifted to 20°C and isopropylgalactoside (IPTG) was added to a final concentration of 0.5 mM. After an additional cultivation for 12 h, cells were harvested and 2.5 ml of buffer A (50 mM TrisHCl, pH 8, 1 M NaCl, 10% glycerol, 10 mM β-mercaptoethanol), containing 0.5 mg/mL lysozyme and 0.5 mM PMSF, was added per gram cells. Disruption of cells was achieved by sonification (Sonifier Generator, Branson) at 4°C and the obtained lysate was centrifuged at 18000 rpm for 60 min. The filtered (0.45 µm) supernatant was then applied to affinity chromatography using an ÄKTAstart™ platform (GE Healthcare) with a 5 mL His-Trap™ HP column (GE Healthcare). The corresponding protein was eluted from the column applying a linear gradient from 0–100 % imidazole (250 mM) in buffer A over 60 min. Collection was conducted by a Frac-30 system (GE Healthcare). Fractions were tested for the presence of the desired protein by SDS-PAGE and CwoeST containing fractions were concentrated by an Amicon Ultra centrifugal filter (Millipore). The purified protein was stored at -80 °C. Assay conditions for CwoeST. The assay conditions for CwoeST were similar to those, previously described for Cpz8.9 The reaction mixture consisted of 0.75 µM CwoeST, 200 µM PAPS, 50 mM sodium phosphate buffer at pH 6.7, 5 mM MgCl2 and 200 μM of different substrates including conexipyrones A-C (1-3), presulficidin B (4), pNP or methylumbelliferone (MU). The reaction solutions were incubated at 30°C for 20 min and reaction was stopped by addition of one volume ice-cold MeOH. The reaction tube was placed on ice for 10 min and centrifuged at 13000 rpm for 10 min. The supernatant was monitored via LC-ESI/MS and MS2 using a Nucleosil 100 C18 column (Dr. Maisch, 3 μm, 100 × 2 mm fitted with a pre- column 10 x 2 mm) at a flow rate of 0.4 mL/min. A linear gradient was applied from 10 to 100 % of solvent B for 15 min (solvent A: 0.1 % formic acid in water; solvent B: 0.06 % formic acid in acetonitrile). For MS analysis, electrospray ionization (alternating positive and negative ionization) in Ultra Scan mode with capillary voltage of 3.5 kV and drying gas temperature of


Page 13 of 23 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


350°C was employed. MS2 analysis was carried out in negative ionization. Product formation was observed at 290 nm and 260 nm. Conditions for two-enzyme assay (CwoeST and Cpz4). The assay conditions for CwoeST and Cpz4 were similar to those, previously described for Cpz8 and Cpz4.9 The reaction mixture (50 µl) consisted of 0.75 µM CwoeST, 1 μM Cpz4, 5 mM MgCl2, 100 μM PAPS, 25 μM hydroxyacylcaprazol E (5) and 100 μM of conexipyrones A, B, C (1-3) or presulficidin B (4). The reaction mixture was incubated at 30°C for 10 min and terminated by adding 50 μL icecold methanol. The tube was placed on ice for 10 min, centrifuged at 13000 rpm for 10 min, and the supernatant finally analyzed at 260 nm via LC-ESI/MS and MS2 as described above.

ASSOCIATED CONTENT Supporting Information Supporting Information Available: This material is available free of charge via the Internet. HR-MS and NMR spectral data for conexipyrones, SDS-PAGE of His8-CwoeST and biochemical investigations, assignment of the sulfation site of caprazamycins and analogues, gBlock and primers.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Orcid-ID Martin C. Konnerth 0000-0002-2988-989X Andreas Kulik

0000-0002-6914-7313

Leonard Kaysser

0000-0002-3943-993X

Harald Gross

0000-0002-0731-821X

Bertolt Gust

0000-0002-1265-0065

Author Contributions Franziska Wiker and Martin Konnerth contributed equally to this work. Funding M.K. is grateful for funding by the DFG-RTG 1708. A.K. was supported by a grant of the SFB 766. 13 ACS Paragon Plus Environment


Page 14 of 23

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank D. Wistuba (Department of Chemistry, Universität Tübingen) for carrying out highresolution MS measurements.

ABBREVIATIONS

REFERENCES (1) Bassler, B. L., and Losick, R. (2006) Bacterially speaking. Cell 125, 237-246. (2) Lerouge, P., Roche, P., Faucher, C., Maillet, F., Truchet, G., Prome, J. C., and Denarie, J. (1990) Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature 344, 781-784. (3) Gangadharam, P. R., Cohn, M. L., and Middlebrook, G. (1963) Infectivity, pothogenicity and sulpholipid fraction of some indian and british strains of tubercle bacilli. Tubercle 44, 452455. (4) Kalan, L., Perry, J., Koteva, K., Thaker, M., and Wright, G. (2013) Glycopeptide sulfation evades resistance. J. Bacteriol. 195, 167-171. (5) Isono, K., Uramoto, M., Kusakabe, H., Kimura, K., Isaki, K., Nelson, C. C., and McCloskey, J. A. (1985) Liposidomycins: novel nucleoside antibiotics which inhibit bacterial peptidoglycan synthesis. J. Antibiot. 38, 1617-1621. (6) Funabashi, M., Baba, S., Nonaka, K., Hosobuchi, M., Fujita, Y., Shibata, T., and Van Lanen, S. G. (2010) The biosynthesis of liposidomycin-like A-90289 antibiotics featuring a new type of sulfotransferase. ChemBioChem. 11, 184-190. (7) Kaysser, L., Siebenberg, S., Kammerer, B., and Gust, B. (2010) Analysis of the liposidomycin gene cluster leads to the identification of new caprazamycin derivatives. ChemBioChem. 11, 191-196. (8) Kobashi, K., Fukaya, Y., Kim, D.-H., Akao, T., and Takebe, S. (1986) A novel type of aryl sulfotransferase obtained from an anaerobic bacterium of human intestine. Arch. Biochem. Biophys. 245, 537-539. (9) Tang, X., Eitel, K., Kaysser, L., Kulik, A., Grond, S., and Gust, B. (2013) A two-step sulfation in antibiotic biosynthesis requires a type III polyketide synthase. Nat. Chem. Biol. 9, 610-615. (10) Chemler, J. A., Buchholz, T. J., Geders, T. W., Akey, D. L., Rath, C. M., Chlipala, G. E., Smith, J. L., and Sherman, D. H. (2012) Biochemical and Structural Characterization of Germicidin Synthase: Analysis of a Type III Polyketide Synthase That Employs Acyl-ACP as a Starter Unit Donor. J. Am. Chem. Soc. 134, 7359-7366. (11) Song, L., Barona-Gomez, F., Corre, C., Xiang, L., Udwary, D. W., Austin, M. B., Noel, J. P., Moore, B. S., and Challis, G. L. (2006) Type III Polyketide Synthase β-Ketoacyl-ACP Starter Unit and Ethylmalonyl-CoA Extender Unit Selectivity Discovered by Streptomyces coelicolor Genome Mining. J. Am. Chem. Soc. 128, 14754-14755. (12) Aoki, Y., Matsumoto, D., Kawaide, H., and Natsume, M. (2011) Physiological role of germicidins in spore germination and hyphal elongation in Streptomyces coelicolor A3(2). J. Antibiot. 64, 607. 14 ACS Paragon Plus Environment

Page 15 of 23 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


(13) Brachmann, A. O., Brameyer, S., Kresovic, D., Hitkova, I., Kopp, Y., Manske, C., Schubert, K., Bode, H. B., and Heermann, R. (2013) Pyrones as bacterial signaling molecules. Nat. Chem. Biol. 9, 573-578. (14) Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403-410. (15) Monciardini, P., Cavaletti, L., Schumann, P., Rohde, M., and Donadio, S. (2003) Conexibacter woesei gen. nov., sp. nov., a novel representative of a deep evolutionary line of descent within the class Actinobacteria. Int. J. Syst. Evol. Microbiol. 53, 569-576. (16) Zhi, X. Y., Li, W. J., and Stackebrandt, E. (2009) An update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int. J. Syst. Evol. Microbiol. 59, 589-608. (17) Kim, Y., Ogura, H., Akasaka, K., Oikawa, T., Matsuura, N., Imada, C., Yasuda, H., and Igarashi, Y. (2014) Nocapyrones: α- and γ-Pyrones from a Marine-Derived Nocardiopsis sp. Mar. Drugs 12, 4110. (18) Oh, D. C., Gontang, E. A., Kauffman, C. A., Jensen, P. R., and Fenical, W. (2008) Salinipyrones and pacificanones, mixed-precursor polyketides from the marine actinomycete Salinispora pacifica. J. Nat. Prod. 71, 570-575. (19) Denoya, C. D., Fedechko, R. W., Hafner, E. W., McArthur, H. A., Morgenstern, M. R., Skinner, D. D., Stutzman-Engwall, K., Wax, R. G., and Wernau, W. C. (1995) A second branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis: its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins. J. Bacteriol. 177, 3504. (20) Michal, G. (1999) Biochemical pathways, Spektrum, Akad. Verlag. (21) Awakawa, T., Crusemann, M., Munguia, J., Ziemert, N., Nizet, V., Fenical, W., and Moore, B. S. (2015) Salinipyrone and Pacificanone Are Biosynthetic By-products of the Rosamicin Polyketide Synthase. ChemBioChem. 16, 1443-1447. (22) Yim, C.-Y., Le, T., Lee, T., Yang, I., Choi, H., Lee, J., Kang, K.-Y., Lee, J., Lim, K.-M., Yee, S.-T., Kang, H., Nam, S.-J., and Fenical, W. (2017) Saccharomonopyrones A–C, New αPyrones from a Marine Sediment-Derived Bacterium Saccharomonospora sp. CNQ-490. Mar. Drugs 15, 239. (23) Kakuta, Y., Pedersen, L. G., Pedersen, L. C., and Negishi, M. (1998) Conserved structural motifs in the sulfotransferase family. Trends Biochem. Sci. 23, 129-130.



Page 16 of 23

Figure Legends: Figure 1. (A) Two-step sulfation process. The type III polyketide synthase Cpz6 is required for biosynthesis of presulficidins (4), which are sulfated by the PAPS-dependent sulfotransferase Cpz8 to yield sulficidins. The PAPS-independent ASST Cpz4 transfers the sulfate group from the sulficidins onto hydroxyaxylcaprazols (5) to yield sulfated hydroxyacylcaprazols (6). (B) Genetic constellation found in Conexibacter woesei DSM 14684 and the respective proposed gene functions according to closest homolog found through BLAST. Figure 2. Production of conexipyrones A-F by heterologous expression of CwoePKS. HPLC profiles of extracts from S. coelicolor M145/Δsco7221/pFW03 (i) and S. coelicolor M145/Δsco7221/pUWL-apra-oriT (ii). Figure 3. MS analysis of feeding studies employing isotope-enriched precursors. Structures of conexipyrones A-C with proposed biosynthetic educts marked in different colors. Mass spectra [M+H]+ of HPLC-ESI/MS analysis of culture extracts from feeding experiments with isotope enriched precursors. Propionate units are depicted in blue, leucine in red, acetate in gold, valine in green and isoleucine in brown. Observed incorporations of labeled carbon atoms, deduced from enhancements of the corresponding mass peaks, are highlighted in the respective colors. Figure 4. Proposed biosynthetic pathway of conexipyrones A-C. Various acyl-CoA starter substrates are iteratively condensed with two extender units of methylmalonyl-CoA. Ring formation is achieved via an intramolecular lactonization reaction. Figure 5. LC-ESI/MS2 analysis of conexipyrone A sulfation by CwoeST. (A) Sulfated conexipyrone A was only identified by extracted ion chromatogram in assays containing PAPS, conexipyrone A and CwoeST. Assays with no CwoeST (B) failed to produce sulfated conexipyrone A. (C) MS/MS fragmentation pattern of the enzymatic reaction products, conexipyrone A. Figure 6. In vitro analysis of a possible two-step sulfation mechanism in Conexibacter woesei using hydroxyacylcaprazol E (5) as a non-genuine acceptor substrate. (A) HPLC analysis of two-enzyme assays demonstrating the sulfation of hydroxyacylcaprazol E (5) resulting in sulfated hydroxyacylcaprazol E (6) with CwoeST, Cpz4, PAPS and presence of 100 µM of either conexipyrone A (1) (i), (1) without CwoeST (ii), (1) without Cpz4 (iii), (1) without PAPS (iv), no conexipyrones (v), conexipyrone B (2) (vi), conexipyrone C (3) (vii) or presulficidin B (4) (viii). UV was measured at 260 nm. (B) Two-step sulfation scheme.


Page 17 of 23 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


66x48mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 1. (A) Two-step sulfation process. The type III polyketide synthase Cpz6 is required for biosynthesis of presulficidins (4), which are sulfated by the PAPS-dependent sulfotransferase Cpz8 to yield sulficidins. The PAPS-independent ASST Cpz4 transfers the sulfate group from the sulficidins onto hydroxyaxylcaprazols (5) to yield sulfated hydroxyacylcaprazols (6). (B) Genetic constellation found in Conexibacter woesei DSM 14684 and the respective proposed gene functions according to closest homolog found through BLAST. 139x95mm (600 x 600 DPI)


Page 18 of 23

Page 19 of 23 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


Figure 2. Production of conexipyrones A-F by heterologous expression of CwoePKS. HPLC profiles of extracts from S. coelicolor M145/Δsco7221/pFW03 (i) and S. coelicolor M145/Δsco7221/pUWL-apra-oriT (ii). 66x71mm (600 x 600 DPI)



Figure 3. MS analysis of feeding studies employing isotope-enriched precursors. Structures of conexipyrones A-C with proposed biosynthetic educts marked in different colors. Mass spectra [M+H]+ of HPLC-ESI/MS analysis of culture extracts from feeding experiments with isotope enriched precursors. Propionate units are depicted in blue, leucine in red, acetate in gold, valine in green and isoleucine in brown. Observed incorporations of labeled carbon atoms, deduced from enhancements of the corresponding mass peaks, are highlighted in the respective colors. 139x118mm (300 x 300 DPI)


Page 20 of 23

Page 21 of 23 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


Figure 4. Proposed biosynthetic pathway of conexipyrones A-C. Various acyl-CoA starter substrates are iteratively condensed with two extender units of methylmalonyl-CoA. Ring formation is achieved via an intramolecular lactonization reaction.

139x76mm (300 x 300 DPI)



Figure 5. LC-ESI/MS2 analysis of conexipyrone A sulfation by CwoeST. (A) Sulfated conexipyrone A was only identified by extracted ion chromatogram in assays containing PAPS, conexipyrone A and CwoeST. Assays with no CwoeST (B) failed to produce sulfated conexipyrone A. (C) MS/MS fragmentation pattern of the enzymatic reaction products, conexipyrone A. 139x78mm (600 x 600 DPI)


Page 22 of 23

Page 23 of 23 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


Figure 6. In vitro analysis of a possible two-step sulfation mechanism in Conexibacter woesei using hydroxyacylcaprazol E (5) as a non-genuine acceptor substrate. (A) HPLC analysis of two-enzyme assays demonstrating the sulfation of hydroxyacylcaprazol E (5) resulting in sulfated hydroxyacylcaprazol E (6) with CwoeST, Cpz4, PAPS and presence of 100 µM of either conexipyrone A (1) (i), (1) without CwoeST (ii), (1) without Cpz4 (iii), (1) without PAPS (iv), no conexipyrones (v), conexipyrone B (2) (vi), conexipyrone C (3) (vii) or presulficidin B (4) (viii). UV was measured at 260 nm. (B) Two-step sulfation scheme. 139x128mm (600 x 600 DPI)


ACS Chemical Biology - ACS Publications - American Chemical Society

Recommend Documents