Studies on Bottromycins. II. Structure Elucidation of Bottromycins B2 and C2.

Kaneda, Miyuki

doi:10.7164/antibiotics.55.924

Cited by 17 publications

(15 citation statements)

References 2 publications

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“…Analysis of the comparative metabolomic and MS 2 network datasets revealed two new molecules (m/z 873.45 and m/ z 887.47) produced by both DbtmF and the amidohydrolase mutant DbtmI,but not by WT S. scabies.Masses of 873.45 Da and 887.47 Da correspond to the addition of H 2 Ot o carboxylated O-desmethylated bottromycins A 2 (1)a nd C 2 , [25] respectively,w hich indicated that one of the cyclodehydrations does not occur in DbtmF and DbtmI.M S n revealed that these molecules are not macrocyclized but do feature the thiazoline ring (10 and 11;F igure 3a;S upporting Information, Figure S10), thus indicating that BtmF and BtmI cooperate to catalyze amidine ring formation, but are not required for thiazoline formation. Both mutant strains also produced ar ange of other bottromycin derivatives that contain at hiazoline ring but no macrocycle ( Figure 1, Figure 2; Supporting Information, Figure S23).…”

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“…An analysis of the metabolomes of DbtmE and DbtmG was used to map molecules produced by these mutants onto this network. Nodes representing species that were not present in DbtmD were manually assessed using MS 2 to identify molecules related to the btm pathway.T his global metabolomic analysis showed that the bottromycin pathway contributes much more to the total metabolite profile of S. scabies than was previously understood, [25] and identified 14 distinct molecules in the wild-type strain, and at least 6a dditional molecules across the mutant strains,w ith masses and fragmentation patterns that are entirely consistent with bottromycin-like molecules (1-20;F igure 1, Figure 2; Supporting Information, Figures S6-S23, Table S3). Theo nly significant molecule that was not revealed by network analysis,owing to al ack of MS 2 fragmentation homology,w as an abundant species with m/z 406.27 (17;F igure 2; Supporting Information, Figure S17), which was identified by the initial comparative analysis of LC-MS data.…”

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Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

et al. 2016

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Bottromycin A 2 is astructurally unique ribosomally synthesized and post-translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrugresistant bacteria. The structural noveltyofbottromycin stems from its unprecedented macrocyclic amidine and rare bmethylated amino acid residues.The N-terminus of aprecursor peptide (BtmD) is converted into bottromycin A 2 by tailoring enzymes encoded in the btm gene cluster.H owever,l ittle was knowna bout key transformations in this pathway,i ncluding the unprecedented macrocyclization. To understand the pathway in detail, an untargeted metabolomic approacht hat harnesses mass spectral networking was used to assess the metabolomes of aseries of pathway mutants.This analysis has yielded key information on the function of av ariety of previously uncharacterized biosynthetic enzymes,i ncluding aY caO domain protein and ap artner protein that together catalyze the macrocyclization.Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products that are prevalent throughout nature, [1] and their biosynthetic pathways are capable of transforming simple proteinogenic amino acids into structurally complex compounds that have potent bioactivities. [2][3][4] However,e lucidating the biosynthesis of RiPPs can be hindered by the difficulty of isolating intermediates,asthe biosynthesis takes place on alarger precursor peptide,a nd intermediates may be rapidly proteolyzed. Therefore,i mproved methods for the identification of RiPP intermediates are desirable.Bottromycin A 2 (1,Scheme 1) [5][6][7][8] possesses potent antibacterial activity towards multidrugresistant bacteria, [9] and is structurally unique owing its unprecedented macrocyclic amidine,r are b-methylated amino acids residues,and aterminal thiazole.Nature employs av ariety of strategies for peptide macrocyclization, [10][11][12] but amidine formation has only been observed for bottromycin. Initial studies on bottromycin biosynthesis showed that its amino acids were b-methylated by radical SAM methyltransferases [5,7] (RSMTs), but the rest of the bottromycin pathway represented abiosynthetic black box, where little was known about key steps in the pathway,including the unprecedented macrocyclization. In this study,weemploy untargeted metabolomics and mass spectral networking to deduce the biosynthetic route to bottromycins in Streptomyces scabies.T his analysis identifies the enzymes responsible for macrocyclization, thiazole formation, and aspartate epimerization, thereby demonstrating the utility of an untargeted metabolomic approach for elucidating atargeted biosynthetic pathway.To assess the role of the putative tailoring genes in the bottromycin pathway (Supporting Information, Figure S1), we had previously generated S. scabies DbtmC, DbtmE, DbtmF, DbtmI,a nd DbtmJ,b ut were unable to identify bottromycin-like compounds in these mutants. [5] We therefore established that these deletions did not lead to deleterious polar effects on the pathway b...

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Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

et al. 2016

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“…The occurrence of a cysteine residue at position 19 in the core peptide region, rather than the expected serine residue usually required for the formation of the aminovinylcysteine, initially led us to think that formation of the aminovinyl-cysteine residue in cypemycin occurred via an unprecedented mechanism. Analysis of the cypD mutant and an in vitro enzyme assay revealed that CypD is solely involved in decarboxylation of the C-terminal cysteine residue (Cys 22 ). Alkylation of the peptide produced by the cypD mutant with iodoacetamide and subsequent ESI analysis confirmed that Cys 19 had been converted into Dha (dehydroalanine).…”

Section: Cypemycinmentioning

confidence: 99%

Understanding and manipulating antibiotic production in actinomycetes

Bibb

2013

Biochemical Society Transactions

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Actinomycetes are prolific producers of natural products with a wide range of biological activities. Many of the compounds that they make (and derivatives thereof) are used extensively in medicine, most notably as clinically important antibiotics, and in agriculture. Moreover, these organisms remain a source of novel and potentially useful molecules, but maximizing their biosynthetic potential requires a better understanding of natural product biosynthesis. Recent developments in genome sequencing have greatly facilitated the identification of natural product biosynthetic gene clusters. In the present article, I summarize the recent contributions of our laboratory in applying genomic technologies to better understand and manipulate natural product biosynthesis in a range of different actinomycetes.

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“…Streptomyces bottropensis strain ATCC 25435 (DSM 40262), obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ), has become an organism of interest due to its ability to produce bottromycin antibiotics (bottromycins A2, B2, and C2), which exhibited activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) (1–3). Herein, we report the draft genome sequence of S. bottropensis ATCC 25435.…”

Section: Genome Announcementmentioning

confidence: 99%