Two Distinct Cyclodipeptide Synthases from a Marine Actinomycete

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Two Distinct Cyclodipeptide Synthases from a Marine Actinomycete Catalyze Biosynthesis of the Same Diketopiperazine Natural Product Elle D. James,† Bryan Knuckley,† Norah Alqahtani,‡ Suheel Porwal,‡ Jisun Ban,† Jonathan A. Karty,§ Rajesh Viswanathan,*,‡ and Amy L. Lane*,† †

Department of Chemistry, University of North Florida, 1 UNF Drive, Jacksonville, Florida 32224, United States Department of Chemistry, Case Western Reserve University, Millis Science Center Room 216, 2074 Adelbert Road, Cleveland, Ohio 44106-7078, United States § Mass Spectrometry Facility, Indiana University, Department of Chemistry, 800 E. Kirkwood Avenue, Bloomington, Indiana 47405, United States ‡

S Supporting Information *

ABSTRACT: Diketopiperazine natural products are structurally diverse and offer many biological activities. Cyclodipeptide synthases (CDPSs) were recently unveiled as a novel enzyme family that employs aminoacyl-tRNAs as substrates for 2,5diketopiperazine assembly. Here, the Nocardiopsis sp. CMB-M0232 genome is predicted to encode two CDPSs, NozA and NcdA. Metabolite profiles from E. coli expressing these genes and assays with purified recombinant enzymes revealed that NozA and NcdA catalyze cyclo(L-Trp-L-Trp) (1) biosynthesis from tryptophanyl-tRNA and do not accept other aromatic aminoacyl-tRNA substrates. Fidelity is uncommon among characterized CDPSs, making NozA and NcdA important CDPS family additions. Further, 1 was previously supported as a biosynthetic precursor of the nocardioazines; the current study suggests that Nocardiopsis sp. may derive this precursor from both NozA and NcdA. This study offers a rare example of a single bacterium encoding multiple phylogenetically distinct enzymes that yield the same secondary metabolite and provides tools for chemoenzymatic syntheses of indole alkaloid diketopiperazines. KEYWORDS: natural product, biosynthesis, diketopiperazine, nonribosomal peptide, cyclodipeptide synthase, tRNA

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that of NRPSs.6 CDPSs divert two aminoacyl-tRNAs (aatRNAs) from their canonical role in ribosomal translation and recruit these as substrates for DKP biogenesis.2,6 Bioinformatics analyses suggest that CDPSs are commonly encoded by bacterial genomes.7 However, fewer than a dozen CDPSs have been functionally characterized,6,8−11 leaving much unknown about this apparently prevalent enzyme family. Nocardioazines A−B are uniquely functionalized diannulated tryptophan DKP natural products from the marine-derived Nocardiopsis sp. CMB-M0232.12 Intermediates relevant to nocardioazine assembly in vivo, beginning with cyclo(L-Trp-LTrp) (1, see Figure 3), were previously supported by comparison of the Nocardiopsis sp. metabolite profile with synthetically generated hypothesized nocardioazine precur-

olecules featuring 2,5-diketopiperazine (DKP) scaffolds are widespread in nature and are structurally diverse.1 The chemical diversity of DKP natural products arises from both the incorporation of a variety of amino acids into these cyclodipeptides as well as extensive enzyme-catalyzed tailoring reactions.1−3 This structural diversity translates into a broad range of biological activities that include antimicrobial, anticancer, and immunosuppressant effects.1 For many years, nonribosomal peptide synthetases (NRPSs) were the sole enzymes recognized as catalysts for DKP assembly.2,3 NRPSs are highly studied, often massive (>100kDa) multimodular enzymes that act as molecular assembly lines to catalyze the formation of peptide bonds between amino acid substrates and modifications to these residues.4,5 In 2009, cyclodipeptide synthases (CDPSs) were first reported as a new family of small (∼30-kDa) enzymes dedicated to DKP assembly through a mechanism fundamentally different from © XXXX American Chemical Society

Received: July 1, 2015

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DOI: 10.1021/acssynbio.5b00120 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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Figure 1. Bioinformatics analyses of CDPSs NozA and NcdA. (a) Organization of genes clustered with nozA and ncdA from Nocardiopsis sp. CMBM0232 and predicted protein functions. The ∼14-kB noz cluster is deposited in GenBank (Accession #KT184400), along with the ∼12-kB ncd cluster (Accession #KT184401). (b) Alignment of NozA and NcdA with all functionally characterized CDPSs.6,8−11 Secondary structure annotations (gray) are based upon features conserved for the structures of crystallized CDPSs AlbC,16 Rv2275,17 and YvmC_lic.10 Residues conserved among all CDPSs are indicated in purple; the conserved active site serine residue is shown in red. For crystallized CDPSs, basic residues within the α4 helix were found to play roles in recognition of the tRNA group of the first aa-tRNA substrate. All characterized CDPSs are enriched with basic residues (shown in dark blue) within this region. Residues in green and yellow comprise the binding pocket for the first and second aminoacyl groups, respectively, of indicated crystallized enzymes. Within these known tRNA and aminoacyl recognition regions, residues distinguishing all three tryptophanyl-tRNA specific CDPSs from promiscuous CDPSs are shown in light blue. (c) Phylogenetic tree of NcdA, NozA, and previously reported CDPSs. The major DKP product is shown with one letter abbreviations for L-amino acids, e.g., cyclo(L-Trp-L-Trp) is listed as cWW. The NvecCDPS2 product is shown as cWX, since multiple amino acids were nonpreferentially incorporated as the second residue of the DKP product.11 The scale bar shows the number of amino acid substitutions per site. Protein accession numbers and all DKP products are listed in Table S3.

sors.13 Heterologous expression of the noz gene cluster (Figure 1a) in Streptomyces coelicolor also suggested 1 as an intermediate in nocardioazine assembly.13 Here, we establish the catalytic function and substrate preferences of NozA and NcdA, CDPS homologues from

Nocardiopsis sp. CMB-M0232. Despite sequence divergence between these two CDPSs, both demonstrate specificity for tryptophanyl-tRNA substrates to yield 1. All but one previously characterized CDPS exhibited promiscuity with respect to aatRNA substrates;9 thus, our finding expands the scope of the B

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Figure 2. HPLC metabolite profiles for E. coli cultures expressing nozA or ncdA. A metabolite with a retention time of 23.7 min was observed for supernatants from E. coli M15 (pREP4) cultures featuring pQE30-nozA or pQE30-ncdA but was absent from cultures with empty pQE30 vector. The retention time of this metabolite matched that of synthetic 1 and ent-1 standard, implicating both NozA and NcdA in the biosynthesis of cyclo(TrpTrp). No cyclo(Trp-Trp) was detected for supernatants from E. coli pQE30-nozA_S36A or pQE30-ncdA_S23A, which produced NozA S36A and NcdA S23A mutants, respectively. Chromatograms show absorbance at 280 nm.

profiles from these cultures were compared to those from cultures carrying empty pQE30 vector by HPLC and LC-MS. These analyses revealed a metabolite with the same retention time from both E. coli pQE30-nozA and pQE30-ncdA cultures but absent from corresponding cultures carrying empty pQE30 vector (Figure 2). No other DKP metabolites distinguished either E. coli pQE30-nozA or pQE30-ncdA cultures from cultures carrying vector only. The biosynthetic product unique to E. coli pQE30-nozA and pQE30-ncdA cultures afforded an [M + H]+ molecular ion at m/z 373, matching that expected for cyclo(Trp-Trp). To conclusively establish the identity of this metabolite, synthetic 1 and ent-1 standards were prepared as reported by Alqahtani et al.13 Briefly, L-Trp or D-Trp was first protected with CbZ chloride and coupled to a second L-Trp or D-Trp unit (preprotected as C-terminal methyl ester) under BOP-Cl conditions. Deprotection and intramolecular cyclization then yielded 1 (from L-Trp) or ent-1 (from D-Trp) in high yields. These synthetic standards exhibited the same C18 HPLC retention time as the biosynthetic product from E. coli pQE30nozA or pQE30-ncdA (Figure 2), supporting these metabolites as cyclo(Trp-Trp). Further indicating both biosynthetic products as cyclo(Trp-Trp), several tandem MS fragments characteristic of synthetic 1 were observed for these metabolites (m/z 242.1, 130.1, 159.1, and 214.1, Figure S1). Finally, highresolution mass spectral data supported that both biosynthetic products featured a molecular formula of C22H20N4O2, consistent with that of 1 ([M + Na]+ m/z 395.1470 from pQE30-nozA; [M + Na]+ m/z 395.1474 from pQE30-ncdA; [M + Na]+ m/z 395.1484 theoretical). To establish the absolute configuration of biosynthetic cyclo(Trp-Trp) from E. coli pQE30-nozA and pQE30-ncdA, purified cyclo(Trp-Trp) from both cultures was hydrolyzed along with synthetic 1 and ent-1 standards. Derivatives of the resulting amino acids were prepared using Marfey’s method.18 LC-MS retention times for derivatives from biosynthetic samples matched those of synthetic 1 standard and differed from those of synthetic ent-1 (Figure S2). These data support that both E. coli pQE30-nozA and pQE30-ncdA yield exclusively 1.

CDPS enzyme family by revealing both NozA and NcdA as uncommon substrate specific CDPSs. Our study also provides a rare example of a bacterium encoding multiple distinct enzymes that yield the same secondary metabolite, and opens doors for application of these enzymes in chemoenzymatic syntheses of molecules occupying the indole alkaloid DKP region of chemical space. Open reading frames (ORFs) were predicted from the draft genome sequence of Nocardiopsis sp. CMB-M0232, and BLASTP14 was employed to determine putative proteins with homology to functionally characterized CDPSs. This revealed two candidate CDPSs, NozA and NcdA. These two CDPS homologues are phylogenetically distinct from one another (53% similarity; Figure 1b,c), suggesting they independently originated from horizontal gene transfer rather than gene duplication within Nocardiopsis sp. CMB-M0232.15 Both nozA and ncdA are clustered with genes that encode proteins homologous to ones playing roles in secondary metabolism (Figure 1a; Tables S1−S2). Genes clustered with nozA are hypothesized to play roles in nocardioazine assembly; precursors relevant to the nocardioazine pathway were proposed in a previous study.13 Of 11 previously characterized CDPSs (Table S3),6,8−11 NozA is most closely related to Ndas_1148 (37% identity; 51% similarity), a promiscuous CDPS that yielded cyclo(L-Phe-LTyr) as the preferred product.8 NcdA shares 67% identity and 78% similarity with Amir_4627, the only previously characterized CDPS exhibiting strict specificity for both aa-tRNA substrates and the only CDPS yielding 1.9 Both NozA and NcdA include all residues strictly conserved among all characterized, catalytically active CDPSs (Figure 1b). Analysis of a phylogenetic tree revealed that CDPSs predominantly cluster into clades that correspond with the type(s) of amino acid(s) incorporated into preferred DKP products (Figure 1c). Intriguingly, NozA diverges from CDPSs that preferentially yield 1. To probe the function of candidate CDPSs NozA and NcdA, corresponding Nocardiopsis sp. CMB-M0232 genes were codon optimized for E. coli expression and cloned. The resulting E. coli M15 (pREP4) pQE30-nozA and pQE30-ncdA cultures were evaluated for DKP production in vivo. Supernatant metabolite C

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Figure 3. In vitro characterization of NozA and NcdA. (a) Both NozA and NcdA catalyze the biosynthesis of 1 from tryptophanyl-tRNA. (b) LCMS2 revealed that the incubation of purified recombinant NozA or NcdA with tryptophanyl-tRNA resulted in the accumulation of 1 relative to no substrate controls. Signals corresponding to an MS2 fragment from the 1 molecular ion (m/z 373 → 242 Da) are shown. To compare titers of 1 for treatments relative to enzyme-only controls, signals are normalized to the analogous MS2 fragment (m/z 401 → 256 Da) from synthetic cyclo-C3methyl-L-Trp-N1′-methyl-L-Trp DKP internal standard (I.S.).

The E. coli pQE30-nozA supernatant yielded 42 ± 5 mg/L of 1 (n = 3, average ± standard deviation), while E. coli pQE30ncdA yielded 11 ± 2 mg/L of 1 (n = 3, average ± standard deviation). The titer of 1 from E. coli pQE30-nozA exceeds that reported from E. coli cultures expressing Amir_4627 (∼30 mg/ L), the sole other CDPS yielding 1. To our knowledge, E. coli pQE30-nozA offers the highest reported yield of 1 from any microbial source. This provides opportunities for enzymatic synthesis of this DKP scaffold, which is a common precursor to several bioactive indole alkaloid DKPs recently targeted by synthetic chemists.1,19,20 Amino acid sequence alignment suggested that both NozA and NcdA include an active site serine residue that is conserved among characterized CDPSs (Figure 1b). Constructs encoding NozA S36A and NcdA S23A were created to probe the function of these hypothesized active site residues. E. coli M15 (pREP4) production of the resulting mutant enzymes was similar to that of the wild type (Figure S3). However, 1 was not detected for supernatants from E. coli pQE-nozA_S36A or E. coli pQE-ncdA_S23A mutant cultures (Figure 2). These data imply that Ser-36 and Ser-23 play an essential role in NozA and NcdA catalysis, respectively. This data suggests that these enzymes employ a catalytic mechanism analogous to that recently proposed for the model CDPS AlbC.21,22 During the first step of AlbC catalysis, the aminoacyl moiety from aa-tRNA is transferred onto the serine active site. This tethered aminoacyl group forms a peptide bond with an aminoacyl moiety donated from a second aa-tRNA substrate. The resulting dipeptidyl intermediate undergoes intramolecular peptide bond formation to close the DKP ring and concomitantly release the DKP product from the active site. To directly probe the function and substrate specificity of NozA and NcdA, each was expressed as a hexahistidine-tagged enzyme in E. coli and purified by Ni affinity and anion exchange chromatography. Interestingly, trace 1 was detected by LC-MS2 for both purified enzymes (Figure 3). This observation suggests that either tryptophanyl-tRNA substrate or 1 strongly binds with NozA and NcdA during purification. To evaluate the

accumulation of 1 for treatments with NozA or NcdA and potential substrates relative to no substrate controls, all samples were spiked with cyclo-C3-methyl-L-Trp-N1′-methyl-L-Trp DKP as internal standard prior to LC-MS2.13 This revealed that 1 accumulated only for treatments in which NozA or NcdA was incubated with tryptophanyl-tRNA (Figure 3), not when enzymes were incubated with tryptophan and/or tRNA (Figure S4). This supports tryptophanyl-tRNA as a substrate of both NozA and NcdA. Both NozA and NcdA are most closely related to CDPSs that catalyze the assembly of DKPs featuring aromatic amino acids (Figure 1c). Thus, the NozA- or NcdA-catalyzed formation of DKPs using other aromatic aa-tRNA substrates was explored. Purified enzymes were incubated with tyrosyl-tRNA, phenylalanyl-tRNA, or combinations of these aa-tRNAs with and without tryptophanyl-tRNA. For treatments without tryptophanyl-tRNA, no product formation was detected by LC-MS. For aa-tRNA substrate mixtures that included tryptophanyltRNA, only 1 was detected (Figure S5). These in vitro data corroborate the in vivo results and establish both NozA and NcdA as CDPSs that catalyze reactions with tryptophanyltRNA, not other proteinogenic aromatic aa-tRNA substrates. Together, our in vivo and in vitro results support that NozA and NcdA are CDPSs that catalyze assembly of the same metabolite, 1 (Figures 2−3). NozA and NcdA are phylogenetically distinct from one another (Figure 1), and thus provide a rare example of two nonduplicate enzymes catalyzing formation of the same secondary metabolite from a single organism. Since Nocardiopsis sp. CMB-M0232 is recalcitrant to genetic manipulation,13 the relevance of nozA and ncdA in the biosynthesis of 1 in vivo was explored by evaluating transcription of these genes during fermentation of Nocardiopsis sp. Reverse transcription PCR (RT-PCR) on days 1−5 of fermentation revealed ncdA transcription on days 2−5, while appreciable nozA transcripts were detected only on day 2 (Figure S6a). These gene transcription patterns largely correlated with the accumulation of 1 in cultures (Figure S6b). Thus, it appears that both NozA and NcdA are relevant in D

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were centrifuged (10 min, 6000 rpm), and supernatants filtered (0.22 μm) and dried in vacuo. The resulting chemical extract was solubilized in H2O (500 μL), and metabolite profiles were compared using HPLC and LC-MS. All experiments were run at least in duplicate. HPLC was completed with a water/acetonitrile gradient (0− 95% over 55 min) using a Zorbax SB-C18 column (Agilent, 4.6 × 150 mm, 5 μm) with a 0.6 mL/min flow rate. LC-MS was conducted with a water/acetonitrile gradient (25−95% over 20 min) using an Acclaim 120 C18 column (Thermo Scientific, 4.6 × 100 mm, 5 μm). ESI+ MS scanning was conducted from m/z 150−1000 using an LTQ-XL (Thermo Scientific) tuned to optimize DKP detection. Cyclo(L-Trp-L-Trp) (1) was purified from 100 mL cultures prepared analogously to those described above. HPLC was completed using the same gradient as above, with a Zorbax SBC18 column (Agilent, 9.4 × 250 mm, 5 μm) and a 3 mL/min flow rate. Fractions containing 1 (23.5−24.0 min) were concentrated in vacuo. Samples were hydrolyzed and Marfey’s method utilized to determine the configuration of resulting amino acids (Supporting Information). Site-Directed Mutagenesis of NozA and NcdA. Following manufacturer directions, the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific) was utilized with primers described in Table S4 to yield site-directed mutants. Constructs were introduced into E. coli M15 (pREP4) and evaluated as described above. Purification of NozA. E. coli M15 (pREP4) pQE30-nozA was prepared and induced with IPTG as described above. Induced cultures were incubated 16 h at 240 rpm and 16 °C. Cells (4 g) were pelleted by centrifugation, resuspended with 16 mL lysis buffer (100 mM Tris HCl pH 8, 150 mM NaCl, 5% glycerol (v/v), 2 mM DTT, 1.25 mg/mL lysozyme), incubated on ice for 30 min, and sonicated. The lysates were centrifuged (12 000 rpm, 4 °C) for 30 min, and the supernatant filtered (0.45 μm). The resulting soluble protein mixture was spiked to 5 mM imidazole and batch bound by incubation with Ni-NTA resin (3 mL, Thermo Scientific) on ice for 30 min. The mixture was poured into a column and eluted sequentially with 1 mL aliquots of lysis buffer (without lysozyme) containing increasing concentrations of imidazole (10 mM, 50 mM, 200 mM, 500 mM). Fractions containing NozA, based on SDSPAGE, were pooled and subjected to further purification by FPLC. FPLC was conducted using a HiTrap Q FF anion exchange column (GE Healthcare). Proteins were eluted with a linear gradient from 100% buffer A (100 mM Tris HCl pH 8, 50 mM NaCl, 5% glycerol, 2 mM DTT) to 100% buffer B (100 mM Tris HCl pH 8, 1 M NaCl, 5% glycerol, 2 mM DTT) over 50 min at a flow rate of 1 mL/min. Fractions containing >90% pure NozA, as evidenced by SDS-PAGE, were concentrated and the buffer was exchanged to assay buffer (50 mM Tris pH 8, 300 mM NaCl) using a 10-kD cutoff centrifugal concentrator. The yield of NozA was ∼3 mg/L. Purification of NcdA. For NcdA purification, synthetic ncdA was ligated into pET30a(+) (Novagen) and introduced into E. coli BL21 (DE3). Cultures were grown in LB with kanamycin (25 μg/mL) at 37 °C with shaking at 200 rpm to an OD630 ∼ 0.4. They were induced with IPTG (1 mM) and incubated at 240 rpm and 19 °C for 16 h. Ni affinity chromatography was conducted as described for NozA. FPLC was conducted as described for NozA, but using a linear elution gradient from 100% buffer A to 30% buffer B. Buffer exchange

biosynthesis of 1 in vivo, and that the nocardioazine pathway may draw 1 from either NozA or NcdA or both. The maintenance and transcription of multiple genes for biosynthesis of 1 suggests that this metabolite or its derivatives play important, yet unknown adaptive roles for Nocardiopsis sp. The apparent specificity of NozA and NcdA for tryptophanyl-tRNA substrates makes them particularly important additions to the CDPS enzyme family. Intriguingly, the only other known substrate specific CDPS, Amir_4627, also employs tryptophanyl-tRNA.9 Although no structural studies have been conducted for substrate specific CDPSs, previous Xray crystallographic studies of promiscuous CDPSs revealed separate binding sites for recognition of each of the two aatRNAs.10,16,17 On the basis of protein alignment, three amino acid residues distinguished tryptophanyl-tRNA specific CDPSs NozA, NcdA, and Amir_4627 from promiscuous CDPSs within these substrate recognition sites. These unique residues included valine and asparagine residues within the basic residue-enriched α4 helix region responsible for recognition of the tRNA group of the first substrate, as well as a basic residue (lysine or arginine) within a site recognized for binding the second aminoacyl group by promiscuous crystallized CDPSs (Figure 1b). Site-directed mutagenesis was completed for these residues of NozA (i.e., V94R/N97A and R203A). Comparison of metabolite profiles between cultures producing wild type and mutant NozA showed no difference in production of 1, and no accumulation of alternate DKP products by LC-MS or HPLC (Figure S7). These data suggest that comparative X-ray crystallographic structures and/or models of tryptophanyl-tRNA specific CDPSs, enabled by our current study, are necessary to unveil features of these CDPSs that confer fidelity.

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METHODS Genome Sequencing and Bioinformatics Analyses. Nocardiopsis sp. CMB-M0232 genome sequencing was conducted as previously described.13,23 Putative ORFs were determined using GeneMark.24 BLASTP14 was employed to find ORFs homologous to characterized CDPSs (Table S3). ClustalW was used for amino acid sequence alignments.25 Phylogenetic trees were created using Geneious TreeBuilder (Biomatters). Synthetic DKP Standards. Cyclo(L-Trp-L-Trp) (1), ent-1, and cyclo-C3-methyl-L-Trp-N1′-methyl-L-Trp DKP standards were prepared as previously reported.13 Other DKP standards (i.e., cFF, cFW, cFY, cWY) were purchased (Bachem). Preparation of Constructs for Expression of nozA and ncdA in E. coli. Genes encoding NozA and NcdA were codon optimized for E. coli expression and synthesized by GeneArt (Life Technologies). Synthetic genes included NdeI and HindIII restriction sites at the 5′ and 3′ ends, respectively (Figure S8). Genes were ligated into the corresponding sites of pQE30 (Qiagen). The resulting pQE30-nozA and pQE30-ncdA constructs were introduced into E. coli M15 (pREP4) for DKP metabolite profiling. Gene insert sequences were confirmed by DNA sequencing. Evaluation of Metabolite Production by E. coli Expressing nozA or ncdA. Cultures (10 mL) of E. coli M15 (pREP4) pQE30-nozA, pQE30-ncdA, and pQE30 were incubated at 200 rpm and 37 °C to an optical density (OD630) of ∼0.4 in LB supplemented with ampicillin (100 μg/mL) and kanamycin (25 μg/mL). Cultures were induced with IPTG (1 mM) and incubated at 240 rpm and 19 °C for 20 h. Cultures E

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(3) Giessen, T. W., and Marahiel, M. A. (2012) Ribosomeindependent biosynthesis of biologically active peptides: Application of synthetic biology to generate structural diversity. FEBS Lett. 586, 2065−2075. (4) Hur, G. H., Vickery, C. R., and Burkart, M. D. (2012) Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology. Nat. Prod. Rep. 29, 1074−1098. (5) Sieber, S. A., and Marahiel, M. A. (2005) Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics. Chem. Rev. 105, 715−738. (6) Gondry, M., Sauguet, L., Belin, P., Thai, R., Amouroux, R., Tellier, C., Tuphile, K., Jacquet, M., Braud, S., Courçon, M., Masson, C., Dubois, S., Lautru, S., Lecoq, A., Hashimoto, S.-i., Genet, R., and Pernodet, J.-L. (2009) Cyclodipeptide synthases are a family of tRNAdependent peptide bond−forming enzymes. Nat. Chem. Biol. 5, 414− 420. (7) Aravind, L., de Souza, R. F., and Iyer, L. M. (2010) Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis. Biol. Direct 5, 48. (8) Giessen, T. W., von Tesmar, A. M., and Marahiel, M. A. (2013) Insights into the Generation of Structural Diversity in a tRNADependent Pathway for Highly Modified Bioactive Cyclic Dipeptides. Chem. Biol. 20, 828−838. (9) Giessen, T. W., von Tesmar, A. M., and Marahiel, M. A. (2013) A tRNA-Dependent Two-Enzyme Pathway for the Generation of Singly and Doubly Methylated Ditryptophan 2,5-Diketopiperazines. Biochemistry 52, 4274−4283. (10) Bonnefond, L., Arai, T., Sakaguchi, Y., Suzuki, T., Ishitani, R., and Nureki, O. (2011) Structural basis for nonribosomal peptide synthesis by an aminoacyl-tRNA synthetase paralog. Proc. Natl. Acad. Sci. U. S. A. 108, 3912−3917. (11) Seguin, J., Moutiez, M., Li, Y., Belin, P., Lecoq, A., Fonvielle, M., Charbonnier, J. B., Pernodet, J. L., and Gondry, M. (2011) Nonribosomal peptide synthesis in animals: the cyclodipeptide synthase of Nematostella. Chem. Biol. 18, 1362−1368. (12) Raju, R., Piggott, A. M., Huang, X.-C., and Capon, R. J. (2011) Nocardioazines: A Novel Bridged Diketopiperazine Scaffold from a Marine-Derived Bacterium Inhibits P-Glycoprotein. Org. Lett. 13, 2770−2773. (13) Alqahtani, N., Porwal, S. K., James, E. D., Bis, D. M., Karty, J. A., Lane, A. L., and Viswanathan, R. (2015) Synergism between Genome Sequencing, Tandem Mass Spectrometry and Bio-Inspired Synthesis Reveals Insights into Nocardioazine B Biogenesis. Org. Biomol. Chem. 13, 7177−7192. (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) Gogarten, J. P., and Townsend, J. P. (2005) Horizontal gene transfer, genome innovation and evolution. Nat. Rev. Microbiol. 3, 679−687. (16) Sauguet, L., Moutiez, M., Li, Y., Belin, P., Seguin, J., Le Du, M. H., Thai, R., Masson, C., Fonvielle, M., Pernodet, J. L., Charbonnier, J. B., and Gondry, M. (2011) Cyclodipeptide synthases, a family of classI aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis. Nucleic Acids Res. 39, 4475−4489. (17) Vetting, M. W., Hegde, S. S., and Blanchard, J. S. (2010) The structure and biosynthesis of the Mycobacterium tuberculosis cyclodityrosine synthetase. Nat. Chem. Biol. 6, 797−799. (18) Marfey, P. (1984) Determination of D-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res. Commun. 49, 591−596. (19) Wang, H., and Reisman, S. E. (2014) Enantioselective total synthesis of (−)-lansai B and (+)-nocardioazines A and B. Angew. Chem., Int. Ed. 53, 6206−6210. (20) Wang, M., Feng, X., Cai, L., Xu, Z., and Ye, T. (2012) Total synthesis and absolute configuration of nocardioazine B. Chem. Commun. 48, 4344−4346. (21) Moutiez, M., Schmitt, E., Seguin, J., Thai, R., Favry, E., Belin, P., Mechulam, Y., and Gondry, M. (2014) Unravelling the mechanism of

was completed as described for NozA. The yield of NcdA was ∼1 mg/L. Characterization of NozA and NcdA Function and Substrate Specificity In Vitro. In vitro characterization of NozA and NcdA was completed using established methods.6 Briefly, E. coli tRNA (50 μM, Sigma #R1753), aminoacyl-tRNA synthetase from E. coli (250 U, Sigma #A3646), amino acid(s) (1 mM of L-Trp, L-Tyr, and/or L-Phe), ATP (5 mM), DTT (2 mM), MgCl2 (10 mM), KCl (30 mM), NaCl (300 mM), and Tris buffer (50 mM, pH 8) were incubated in a total volume of 100 μL for 20 min at 37 °C to yield aa-tRNAs. Purified NozA or NcdA (∼5 μM) was then added to the reaction mixture and incubation continued at 37 °C overnight. Reactions were quenched with TCA (5 μL, 100% w/v), supplemented with cyclo-C3-methyl-L-Trp-N1′-methyl-L-Trp DKP internal standard (1 μM), centrifuged (13 000 rpm, 30 min), and the supernatant subjected to LC-MS as described above. Assays were conducted at least in duplicate. Control assays were conducted by omitting tRNA, amino acid(s), NozA or NcdA, and/or aminoacyl-tRNA synthetase.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.5b00120. Additional methods; ncd and noz cluster annotation; overview of characterized CDPSs; SDS-PAGE protein profiles; primer sequences; tandem MS; Marfey’s analysis results; additional LC-MS2 in vitro assay data; RT-PCR results; metabolite profiles for NozA aa-tRNA binding site mutants; synthetic gene sequences. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Robert Capon (University of Queensland) for generously sharing Nocardiopsis sp. CMB-M0232. A.L.L. is grateful for funding from a Research Corporation Cottrell College Science Award, University of North Florida (UNF) Academic Affairs grants, and a Dean’s Leadership Council Faculty Fellowship. E.D.J. was supported by a UNF Biology Graduate Summer Research Award. Portions of this study were supported by funding from Case Western Reserve University. N.A. was supported by a fellowship from the Saudi Arabian Cultural Mission. R.V. is grateful for funding from Case Western Reserve University’s Start-Up award and support from the Dean’s Office for Inclusion Diversity and Excellence achievement award.

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REFERENCES

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