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Structure Elucidation and Biosynthesis of Fuscachelins, Peptide Siderophores from the Moderate Thermophile Thermobifida Fusca

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Structure Elucidation and Biosynthesis of Fuscachelins, Peptide Siderophores from the Moderate Thermophile Thermobifida Fusca Structure elucidation and biosynthesis of fuscachelins, peptide siderophores from the moderate thermophile Thermobifida fusca Eric J. Dimise, Paul F. Widboom, and Steven D. Bruner* Department of Chemistry, Boston College, Eugene F. Merkert Chemistry Center, Chestnut Hill, MA 02467 Edited by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved August 20, 2008 (received for review June 4, 2008) Bacteria belonging to the order Actinomycetales have proven to be an biosynthetic genes with the product, prediction of the peptide important source of biologically active and often therapeutically structure is possible. Despite several recent examples of this ap- useful natural products. The characterization of orphan biosynthetic proach, there remain aspects of NRP biosynthesis that are difficult gene clusters is an emerging and valuable approach to the discovery to predict, including uncommon amino acid incorporation, domain of novel small molecules. Analysis of the recently sequenced genome skipping/repeating, and macrocyclization. of the thermophilic actinomycete Thermobifida fusca revealed an Thermobifida fusca is a moderately thermophilic actinomycete orphan nonribosomal peptide biosynthetic gene cluster coding for an widely studied as a model organism for thermostable extracellular unknown siderophore natural product. T. fusca is a model organism cellulases (16–20). The genomic sequence of T. fusca YX was for the study of thermostable cellulases and is a major degrader of reported recently (21). There are no characterized secondary plant cell walls. Here, we report the isolation and structure elucidation metabolites from this actinomycete and few characterized natural of the fuscachelins, siderophore natural products produced by T. products from any thermophilic bacteria or archaea (22). One fusca. In addition, we report the purification and biochemical char- recent example is the elucidation of benzodiazepine biosynthesis in acterization of the termination module of the nonribosomal peptide Streptomyces refuineus (23). Here, we describe a family of structur- synthetase. Biochemical analysis of adenylation domain specificity ally novel nonribosomal peptide siderophores, termed fuscachelins, supports the assignment of this gene cluster as the producer of the produced by an orphan gene cluster from T. fusca. The elucidated BIOCHEMISTRY fuscachelin siderophores. The proposed nonribosomal peptide bio- biosynthetic pathway contains many unusual aspects that were not synthetic pathway exhibits several atypical features, including a predictable by bioinformatic analysis. In addition, structure eluci- macrocyclizing thioesterase that produces a 10-membered cyclic dep- dation of the fuscachelins revealed a molecular architecture not sipeptide and a nonlinear assembly line, resulting in the unique observed in iron-chelating siderophore secondary metabolites. heterodimeric architecture of the siderophore natural product. Results ͉ ͉ natural product isolation nonribosomal peptide biosynthesis Siderophore Gene Cluster in T. fusca. An uncharacterized gene genome mining cluster in T. fusca contains genes corresponding to a multimodular NRP synthetase secondary metabolite biosynthetic pathway [see ron is a nutrient that is required by virtually all organisms to supporting information (SI) Fig. S1]. Three NRP synthetase genes Iconduct essential life processes. Under aerobic conditions, the designated fscGHI are contiguous in the cluster and correspond to ferric oxidation state predominates as the extraordinarily water- five peptide extension modules (Fig. 1A). The first gene, fscG, insoluble Fe(OH)3 salt (1). These environmentally limiting condi- encodes a 390-kDa protein comprising three NRP synthetase tions have placed selective pressure on organisms to develop modules, each containing the core elongation domains: condensa- controlled and specific mechanisms to acquire iron. Siderophores tion (C), adenylation (A), and peptidyl carrier protein (PCP). The are secondary metabolites used to scavenge ferric ion selectively first module contains a predicted epimerization (E) domain, sug- through the formation of soluble chelation complexes (2, 3). This gesting that the stereochemistry of the corresponding amino acid in structurally diverse group of small molecules contains metal- the product has the D-configuration. The second gene, fscH, chelating motifs that commonly include hydroxamates, catechols, contains a single elongation module and is followed by a gene for ␣-hydroxyacids, and heterocycles to bind iron with high affinity. the termination module, FscI, which contains a C-terminal thioes- Iron uptake is frequently a limiting factor for growth, including in terase (TE) domain. Upstream of fscGHI are genes with sequence human hosts, making siderophores virulence factors in a variety of homology to characterized 2,3-dihydroxybenzoic acid (Dhb) bio- human pathogens and a target for antimicrobial therapy (4–6). synthetic genes. FscA, FscB, and FscD are homologous to the well The constantly expanding pool of microbial genomic sequence characterized catecholate biosynthetic enzymes: isochorismate syn- data has prompted the isolation of new natural products through thase, isochorismatase, and 2,3-dihydro-Dhb dehydrogenase (24– the identification of orphan biosynthetic gene clusters (7–9). Ex- 26). An adenylation domain (FscC) with predicted specificity for ploiting the predictive nature of biosynthetic pathways, natural Dhb and a dedicated aryl-carrier protein (FscF) are present as products can be isolated and characterized by using an assay-guided stand-alone domains to incorporate Dhb as the starter building fractionation approach. Nonribosomal peptide (NRP) biosynthetic block. Additional proximal genes are present that are homologous machinery is often used to construct siderophores (10, 11). Peptide- based architectures allow for the incorporation of common iron- chelating functionalities. Structure elucidation of nonribosomal Author contributions: E.J.D., P.F.W., and S.D.B. designed research; E.J.D. and P.F.W. per- peptide natural products is particularly amenable to a genome formed research; E.J.D., P.F.W., and S.D.B. analyzed data; and E.J.D., P.F.W., and S.D.B. mining approach because the assembly-line nature of the enzymatic wrote the paper. machinery leads to predictable products. NRP synthetases are The authors declare no conflict of interest. large, multidomain enzymes catalyzing the assembly of peptides by This article is a PNAS Direct Submission. a thioester-templated mechanism (10). Identification of amino acid *To whom correspondence should be addressed. E-mail: [email protected]. building blocks is possible from analysis of the sequence of the NRP This article contains supporting information online at www.pnas.org/cgi/content/full/ synthetase adenylation domains (12–15). By using this information 0805451105/DCSupplemental. in combination with the frequently observed colinearity of the © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805451105 PNAS ͉ October 7, 2008 ͉ vol. 105 ͉ no. 40 ͉ 15311–15316 Downloaded by guest on September 26, 2021 sponding to fuscachelin B) was further purified by reverse-phase chromatography, and the structure was determined. 1H, TOCSY, and COSY experiments established the amino acid content of the peptide by identifying the individual amino acid spin systems: Arg, Gly, Ser, HOOrn in addition to Dhb (Figs. S3 and S4). Unexpect- edly, 13C and 15N gHMBC experiments to confirm the amino acid connectivity revealed a heterodimeric peptide with the sequence Dhb-Arg-Gly-Gly-Ser-HOOrn-Gly-Gly-Arg-Dhb (Table 1 and Fig. S5). The mass of the isolated product supports this structure with a measured exact m/z of 1048.4448 ([MϩH]ϩ, calculated 1048.4448) and a molecular formula of C42H62N15O17 (Fig. S6). Close exam- ination of the 1H NMR spectra reflects a heterodimeric structure. For example, the integration of the amide and ␣-C protons is 2:4:1:1, Arg:Gly:Ser:HOOrn (see Fig. S3), and fine double signals are evident in the asymmetric halves of the molecule (for example, the protons of Arg and Dhb). To confirm the NMR structural assignment, MALDI-TOF/TOF fragmentation (Fig. 3 and Fig. S6) was performed, and all fragments are consistent with the predicted structure. The determination of amino acid chirality was conducted by using Marfey’s method (36), indicating the presence of D-Arg, Gly, L-Ser, and L-HOOrn in a Ϸ2:4:1:1 ratio based on peak integration (Fig. S7). An additional chromatographic peak from the T. fusca preparation, eluting just before fuscachelin B, exhibited NMR spectra very similar to fuscachelin B. The measured m/z of this distinct product was 1047.4614 ([MϩH]ϩ, calculated 1047.4608, C42H63N16O16) corresponding to a change to an NH versus an O in fuscachelin B. Inspection of the 1H NMR and mass spectral fragmentation data showed that this difference was localized at the HOOrn residue, and additional NMR experiments, in particular 1H/15N gHSQC, are consistent with an ␣-amide structure (termed Fig. 1. The predicted gene products of a nonribosomal peptide biosynthetic fuscachelin C) as shown in Fig. 2B (Fig. S8). cluster in T. fusca.(A) Schematic organization of the nonribosomal peptide A third CAS-positive fraction (fuscachelin A) was evident in the assembly line components. (C, condensation; A, adenylation; ArCP, aryl carrier protein; E, epimerization; PCP, peptidyl carrier protein; and TE, thioesterase chromatographic separation (see Fig. 1A). This
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