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Nov 7, 2017 - Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States. ‡. Chemical Biology Institute, Yale University,...
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Functional Characterization of a Condensation Domain that Links Nonribosomal Peptide and Pteridine Biosynthetic Machineries in Photorhabdus luminescens Corey E Perez, Hyun Bong Park, and Jason M. Crawford Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00863 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 7, 2017

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Functional Characterization of a Condensation Domain that Links Nonribosomal Peptide and Pteridine Biosynthetic Machineries in Photorhabdus luminescens Corey E. Perez,‡, § Hyun Bong Park,‡, § and Jason M. Crawford*,‡,§,∥ ‡

Department of Chemistry, Yale University, New Haven, CT 06520, USA Chemical Biology Institute, Yale University, West Haven, CT 06516, USA ∥Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06510, USA *Correspondence: [email protected] §

Abstract. Nonribosomal peptide synthetases (NRPSs) produce a wide variety of biologically important small molecules. NRPSs can interface with other enzymes to form hybrid biosynthetic systems that expand the structural and functional diversity of their products. The pepteridines are metabolites encoded by an unprecedented pteridine-NRPS-type hybrid biosynthetic gene cluster in Photorhabdus luminescens, but how the distinct enzymatic systems interface to produce these molecules has not been examined at the biochemical level. By an unknown mechanism, the genetic locus can also affect the regulation of other enzymes involved in autoinducer and secondary metabolite biosynthesis. Here, through in vitro protein biochemical assays, we demonstrate that an atypical NRPS condensation (C) domain present in the pathway condenses acyl-units derived from α-keto acids onto a free 5,6,7,8-tetrahydropterin core producing the tertiary cis-amide-containing pepteridines. Solution studies of the chemically synthesized molecules led to the same amide regiochemistries as those observed in the natural products. The biochemical transformations mediated by the C domain destroy the radical scavenging activity of its redox active tetrahydropterin substrate. Secondary metabolite analyses revealed that the pepteridine locus affects select metabolic pathways associated with quorum sensing, antibiosis, and symbiosis. Taken together, the results suggest that the pathway likely regulates cellular redox and specialized metabolic pathways through engagement with the citric acid cycle. Introduction. Nonribosomal peptides represent a diverse class of specialized metabolites that often exhibit antimicrobial, anticancer, and immunomodulatory activities, among others (1, 2). They are constructed by nonribosomal peptide synthetases (NRPSs), which are capable of using a wide variety of both proteinogenic and nonproteinogenic amino acid substrates (3-5). A minimal canonical NRPS extender module (C-A-T) contains an adenylation (A) domain responsible for the selection and activation of its cognate substrate; a thiolation (T) domain, a carrier protein that harbors a post-translationally appended phosphopantetheinyl arm responsible for covalently binding and ferrying the nascent peptidyl products; and a condensation (C) domain that catalyzes the union of upstream and downstream carrier proteintethered building blocks, primarily through trans-amide bond formation (6). The carrier protein is shared among fatty acid, nonribosomal peptide, and polyketide biosynthetic systems, and consequently, combinatorial pathways comprised of these systems represent a common strategy for metabolite structural diversification (7, 8). The biocatalytic capabilities of NRPS C domains can further be expanded by condensing “free” substrates, such as nucleosides in the production of nucleoside antibiotics and carbohydrates/aminoglycosides in the production of glycopeptide antibiotics, among others (9, 10). Genome sequencing and bioinformatic analyses of isolates of the gammaproteobacterial entomopathogen Photorhabdus luminescens have revealed that its genome encodes an expansive set of biosynthetic pathways to produce a myriad of specialized metabolites (11, 12). Photorhabdus is harbored in the gut of its mutualistic Heterorhabditis nematode host, and together they migrate, find, and infect insect larvae. P. luminescens exhibits phenotypic variation in their shared pathogenic lifestyle. The bacterium’s primary phenotypic form (the P-form) is highly virulent in insect larvae relative to its small colony variant form (M-form), which has alternatively been associated with the colonization of specific intestinal cells in its nematode host (13-17). It is during active infections in their pathogenic P-form that P. luminescens predominately activates its secondary biosynthetic machinery producing a ACS Paragon Plus Environment

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diverse set of molecules which serve as antimicrobials, immunosuppresants, and nematode development signals, among other roles (18, 19). Select Photorhabdus species can also cause human infections (20-22).

Figure 1. The Pepteridine Biosynthetic Gene Cluster and Key NRPS-Pteridine Coupling Reaction. The biosynthetic gene cluster responsible for the formation of pepteridine A (1) and B (2) is shown. Green genetic regions denote pteridine synthesis machinery, blue genetic regions denote pyruvate dehydrogenase-like machinery, and red genetic regions highlight NRPS-like regions. The predicted function of each gene is noted – T, thiolation; and C, condensation. The inset represents the predicted biosynthesis in which free tetrahydropterin is functionalized with an acyl appendage present on the phosphopantetheinyl arm of the T domain through the action of the C domain. The known products of the pathway, pepteridine A (1) and B (2) are shown (metabolite color codes correspond to associated genetic regions in the pathway). We recently reported the characterization of a hybrid biosynthetic pathway in P. luminescens that invokes pteridine biosynthesis, pyruvate dehydrogenase-like biochemistry, and NRPS-type domains (T-C) (Figure 1) to produce the small molecule metabolites pepteridine A (1) and B (2) (23). The pepteridines feature a tetrahydropterin core functionalized at its N5-position with a varying acyl moiety. Using genetically locked phenotypic variants of P. luminescens, we previously demonstrated that the pepteridines were detected only in the P-form phenotypic variant. Through quantitative proteomics, we also showed that the genetic locus positively affects pyrone quorum sensing and other specialized metabolic enzymes when a LysR-type transcriptional repressor (hexA) (24, 25) associated with the mutualist/pathogen transition is derepressed (23). Notably, the Plu2796 enzyme located in the genomic island shares homology with the pyruvate dehydrogenase E2-like subunit and with NRPS T and C domains. Rather than converting pyruvate to acetyl-CoA as in the well-known pyruvate dehydrogenase reaction, we proposed that pyruvate and α-ketobutyrate were oxidatively decarboxylated and directly converted to acyl-T rather than the canonical acyl-CoA products (23). This type of carrier protein loading has also been proposed in the biosyntheses of select N-acyl-amides (26), the pristinamycin IIa streptogramin antibiotic (27), naphthyridinomycin antitumor antibiotics (28), and the RNA polymerase inhibitor ripostatin (29). α-Ketobutyrate and pyruvate feeding studies in conjunction with stable isotopic labeling studies supported this hypothesis for the pepteridines (23). Site-directed inactivation of the T domain destroyed pepteridine production in bacterial culture, supporting that the resulting acyl-substrates are indeed carrier protein bound (23). Based on protein homologies and bioinformatic analysis, we further proposed that the atypical C domain condenses these acyl-loaded carrier protein substrates with a free tetrahydropterin substrate to generate the tertiary cis-amide bond observed in the products, but this hypothesis has not been experimentally tested. ACS Paragon Plus Environment

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Here, utilizing an isolated C monodomain construct we recapitulate the production of the pepteridines in vitro and confirm their structures via synthesis and co-elution studies. Our study, which focuses on the key amide bond formation step in pepteridine biosynthesis, provides the first biochemical characterization of a NRPS-pteridine hybrid pathway. The biological relevance of the pepteridine genetic locus was further investigated using metabolomic analysis of a locus deletion construct and in vitro assays. Materials and Methods. General. An Agilent 6120 quadrupole liquid chromatography/mass spectrometry (LC/MS) system (Agilent Technologies, USA) fitted with a Phenomenex Kinetex C18 (100 Å) 5 µm (4.6 × 250 mm) column (Phenomenex, USA) was used to acquire low-resolution electrospray ionization mass spectrometry (ESI-MS) data. High-resolution (HR) ESI-MS data were acquired using an Agilent iFunnel 6550 QTOF (quadrupole time-of-flight) MS coupled to an Agilent 1290 Infinity HPLC system. Preparative HPLC was carried out on an Agilent PrepStar system fitted with a 1260 Infinity DAD VL. Proton NMR spectra were acquired at 600 MHz on an Agilent NMR spectrometer equipped with a cold probe, while 13C NMR spectra were recorded at 100 MHz on an Agilent NMR spectrometer. An EnVision Multilabel Plate Reader (PerkinElmer, USA) was utilized for quantification of microtiter plate assays. Photorhabdus luminescens TTO1 (P. luminescens TTO1) was maintained on lysogeny broth (LB) agar plates. When needed, suspension cultures were generated by selecting single, well-defined colonies into LB medium followed by overnight outgrowth at 30 °C and 250 rpm. E. coli strains were similarly maintained, but cultivations were performed at 37 °C. When needed, kanamycin (American Bioanalytical, USA) was used at 25 µg/mL. Cloning and Expression of the Plu2796 C Monodomain. Standard molecular biology (pET28a, EMD Biosciences), protein expression (E. coli BAP1), and protein purification (Ni-NTA resin, Qiagen) procedures were employed in preparing an N-terminal His6-tagged variant of the Plu2796 C domain (Plu2796-C) for biochemical analysis (see supporting information). In vitro Activity Analysis of Plu2796-C. Pterin-containing molecules (pterin, 6-biopterin, D-(+)neopterin, and tetrahydrofolic acid) and acyl-CoAs (acetyl-, propionyl-, malonyl-, and succinyl-CoA) were purchased from Millipore Sigma. The pterin substrates (0.25 mmol) were reduced via platinum oxide catalyzed hydrogenation (5.7 mg, 0.025 mmol, Millipore Sigma) in anhydrous methanol. Enzyme reactions were carried out at a 50 µL scale in triplicate and contained 400 µM acyl-CoA, 1 µL of the freshly prepared tetrahydropterin solution, and 10 µM Plu2796-C in a reaction buffer consisting of 50 mM NaH2PO4, pH = 7, and 2 mM tris(2-carboxyethyl)phosphine (TCEP). All solutions were sparged with nitrogen. The reaction mixtures were sealed under nitrogen and incubated at 25.0 °C for 1 hr before being quenched with butanol. The dried butanolic extracts were subsequently analyzed via reversed-phase liquid chromatography/high-resolution quadrupole time-of-flight mass spectrometry (LC/HR-QTOF-MS) (see supporting information). Synthesis of Pepteridine A and B. Pterin 4 (1.0 g, 6.13 mmol) was suspended in anhydrous pyridine (40 mL). Isobutyric anhydride (10 mL, 60.31 mmol) was added to the pterin suspension, and the reaction was refluxed for 5 h. The reaction mixture was dried and purified by several rounds of reversed-phase liquid chromatography to yield 4a (tR = 24 min, 1.1 g, 76.9%). Hydrogenation of compound 4a (1.1 g, 4.72 mmol) in methanol (100 mL) was performed utilizing platinum oxide (0.1 g, 0.44 mmol) as a catalyst. After 24 h, the resulting materials were filtered through Celite545 (Millipore Sigma, USA), and the filtrates were evaporated under reduced pressure. Reversed-phase purification of the reaction mixture yielded 4b (tR = 14 min, 0.8 g, 71.5%). Compound 4b (0.4 g, 1.69 mmol, each) was subsequently added to two different vials and dissolved in anhydrous pyridine (30 mL) under purging with nitrogen gas. The N-acylation reactions were initiated by adding propionyl chloride (1.0 equiv) or acetyl chloride (1.0 equiv), respectively, and were then stirred under nitrogen gas for 10 min before being quenched by the addition of water. Reversed-phase separation of the dried reaction mixture led to compound 4c (tR = 22 min, 0.27 g, 54.5%) and compound 4d (tR = 18 min, 0.25 g, 53.0%). Compounds 4c (0.2 g, 0.68 mmol) and 4d (0.2 g, 0.72 mmol) were dissolved in anhydrous methanol (20 mL), and sodium methoxide (0.012 g, 0.22 mmol) was added into the individual reaction ACS Paragon Plus Environment

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vials. After 4 h, the reaction mixtures were dried and subjected to reversed-phase chromatography to afford compound 1 (tR = 25.6 min, 0.12 g, 79.1%) and 2 (tR = 20.3 min, 0.12 g, 79.7%), respectively. Radical Scavenging Activity. A nitrogen-containing glove box was utilized for all experimental steps. The standard di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) radical scavenging assay was employed with minor modifications. Tetrahydropterin was freshly prepared via hydrogenation of commercial pterin (Millipore Sigma) as described above. Tetrahydropterin, pepteridine A, pepteridine B, and L-ascorbic acid were resuspended at 20 mM in anhydrous, nitrogen-sparged DMSO, and two-fold serially diluted. 200 µL reactions containing 0.1 mM DPPH were prepared with compounds ranging in concentrations tested from 1 mM to 0.49 µM. The reactions were allowed to proceed in the dark for 30 min and were subsequently quantified via absorbance at 517 nm. Half-maximal inhibitory concentrations (IC50) were calculated using GraphPad Prism (v 7.03, GraphPad Software, USA). Assessment of Metabolite Production. Five single, well-defined colonies of P. luminescens TTO1 ∆hexA and P. luminescnes TTO1 ∆hexA/locus from two day growths on LB agar plates were selected into LB for overnight growth. The outgrowths were subcultured into 5 mL LB and allowed to grow for two days at 30 °C and 250 rpm. Extraction and analysis protocols were conducted as previously reported (23). Briefly, the supernatants were clarified via centrifugation and subsequently extracted with 6 mL water-saturated butanol. 5 mL of the butanolic layers were dried via centrifugal evaporation, and the dried extracts were resuspended in 200 µL methanol for LC/HR-QTOF-MS. Results and Discussion. To determine whether the C domain catalyzes the key amide bond formation step in pepteridine biosynthesis, we cloned and isolated the Plu2796 C monodomain (Plu2796-C, Figure S1, Table S1) as an N-terminal His6-tagged variant for biochemical analysis. We used acyl-CoA substrates (acetyl-, malonyl-, propionyl-, and succinyl-CoA) as upstream acyl-phosphopantetheinyl-T domain mimetics to examine C-domain acyl-selectivity. Common branched chain acyl-CoAs were not utilized in this study as branched-chain pepteridines were not detected under heterologous expression conditions nor in the native P. luminescens host (with and without branched-chain α-keto acid supplementations) (23). As the C domain was hypothesized to utilize free pteridine substrates, pterin, biopterin, neopterin, and tetrahydrofolate, all biologically-relevant pteridines that P. luminescens could be exposed to during its lifecycle, were chosen to examine pterin substrate selectivity. In vitro biochemical reactions were prepared in triplicate containing the appropriate acyl-CoA, tetrahydropteridine, and 10 µM of the Plu2796 C monodomain in a sodium phosphate buffer system. End-point reactions were performed at 25.0 °C under a nitrogen atmosphere for 1 hour before being quenched with butanol. The dried butanolic extracts were analyzed by LC/HR-QTOF-MS (Figures 2 and S2). Of the pterin substrates, the C domain only utilized 5,6,7,8-tetrahydropterin, as observed in the natural products. C6functionalization, a conserved structural feature of known pterin cofactors, destroyed activity. The acylsubstrate selectivity was largely in agreement with the pepteridine products observed in bacterial cultures with acetyl-, propionyl-, and malonyl- units being accepted by the C domain. We were previously not able to detect malonyl-functionalized product 3 (pepteridine C) in P. luminescens nor in E. coli cultures overexpressing the pathway (23). This suggests that either the α-keto acid oxaloacetate is not a substrate for the upstream E1 dehydrogenase in vivo or the malonyl-product decarboxylates under the conditions of our cultivation and extraction experiments, leading to the enhancement of 2. Taken together, the acyl- and pterin substrate selectivities in vitro leading to enzymatic products 1–3 are consistent with the observation of metabolites 1 and 2 in vivo.

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Figure 2. In vitro Biochemical Assessment of Plu2796-C Activities. Extracted ion chromatograms within 10 ppm of the calculated exact monoisotopic protonated mass for each molecule are shown for each experimental condition. 1–3 have been confirmed by co-elution studies using synthetic standards. To confirm the pepteridine structures and to assess their in vitro activities in a panel of assays, we synthesized 1, 2, and 3 employing a synthetic scheme analogous to published procedures for N5acylated tetrahydropterin (Scheme 1, Figures S3-12) (30, 31). Briefly, N2-isobutyroylpterin was prepared via the addition of isobutyl anhydride to pterin (4). PtO2-catalyzed hydrogenation of N2isobutyroylpterin yielded the N2-isobutyroyl-5,6,7,8-tetrahydropterin intermediate, which was subsequently reacted with the appropriate acyl chloride. Finally, deprotection with sodium methoxide in methanol yielded the final products. Comparative NMR and LC/MS data analyses of the natural and synthetic materials unambiguously confirmed the structures of the natural metabolites (Figures S13S15). The tertiary cis-amide regiochemistries noted in the natural products were similarly observed in solution (DMSO-d6) NMR studies of the synthetic products. Additionally, co-elution studies using synthetic 1, 2, and 3 confirmed the identity of the enzymatic products (Figure S16).

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Scheme 1. Synthesis of Pepteridine A (1) and B (2). Reagents and conditions: (a) isobutyric anhydride, pyridine, reflux, 5 h, 76.9%; (b) PtO2, H2, rt, 24 h, 71.5%; (c) propionyl chloride for 4c, rt, 10 min, 54.5%; acetyl chloride for 4d, rt, 10 min, 53.0%; (d) MeOH, CH3ONa, rt, 4 h, 79.1% for 1 and 79.7% for 2. See supporting information for synthesis of enzymatic product 3. The functional relevance of pepteridine formation can be rationalized by examining known pterin cofactor systems. The mechanism of action of pterin cofactor-dependent hydroxylases is believed to require the reaction of a tetrahydropterin C4a with molecular oxygen passing through a cationic radical intermediate (32). Similarly, in the mechanism of action of nitric oxide synthase, the tetrahydrobiopterin cofactor is invoked as a one-electron donor (33). Various studies of the pterin radical state demonstrate that the majority of the unpaired electron spin density lies on N5 (34, 35). Indeed, N5-alkylation inhibits the ability of tetrahydropterins to serve as competent cofactors for tyrosine hydroxylase (36). Given the radical chemistry known for tetrahydropterins (32), we hypothesized that N5-acyl-functionalization could remove free pterin from the cellular redox pool. Consequently, we assessed the redox activity of 1 and 2 and their precursor 5,6,7,8-tetrahydropterin via a radical scavenging assay using the stable free radical indicator di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) (Figure 3). Both an L-ascorbic acid control and 5,6,7,8-tetrahydropterin demonstrated nearly complete radical scavenging activity at and above 62.5 µM, with 5,6,7,8-tetrahydropterin (IC50 = 23 µM) being slightly more potent than Lascorbic acid (IC50 = 26 µM) at lower concentrations (Figure 3). Despite the tetrahydro-core being present in both pepteridines, neither 1 nor 2 demonstrated radical scavenging activity at concentrations up to 1 mM (Figure 3). As anticipated, this suggests that N5-acylation inhibits the formation of the quinonoid state, a critical intermediate during the oxidation cascade (32). As such, Plu2796-mediated N5-acylation appears to catalyze the formation of “blocked” metabolites that remove free tetrahydropterin from the cellular redox pool. At the mechanistic level, it remains unclear how the pepteridine substrates/products or the redox-relevant biochemical process itself contribute to the modulation of pyrone quorum sensing.

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Figure 3. Radical Scavenging Activities. The ability of pepteridine A (1), pepteridine B (2), and 5,6,7,8tetrahydropterin to quench the stable free radical di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) indicator is shown. L-Ascorbic acid and tetrahydropterin exhibit half-maximal inhibitory concentrations (IC50) of 26 and 23 µM, respectively. As the pepteridines are “redox-blocked” substrates that share structural similarities to known pterin based cofactors (32, 37-39), we further subjected the pepteridines to a series of antimicrobial, anticancer, and enzyme inhibitory assays. The molecules failed to significantly inhibit the growth of selected microbial strains (Candida albicans, Saccharomyces cerevisiae, Escherichia coli Nissle 1917, and Bacillus subtilis; Figure S17). At 10 µM, both 1 and 2 possessed weak cytotoxic activities against a colon cancer cell line (HCT-116, 77% and 79% mean growth percent for (1) and (2), respectively, relative to vehicle controls set at 100%), whereas only 2 exhibited weak activity against a breast cancer cell line (T-47D, 77% mean growth percent) in the NCI-60 human tumor cell line anticancer drugscreening program (Figures S18-S19). Functionalized pteridines have been reported to have kinase inhibitory activities (40-42), so we also evaluated the pepteridines in a panel of human kinases. Pepteridine 1 exhibited some inhibitory activity at 10 µM against the TBK1 kinase (65 ± 3% activity, Table S2, negative controls set at 100% activity). Consistent with the lack of both redox and appreciable growth inhibitory activities, 1 and 2 also lacked dihydrofolate reductase, nitic oxide synthase, and xanthine oxidase inhibitory activities (Figures S20-S22). While we demonstrated here that pepteridine formation eliminates pterin-mediated radical scavenging activity in vitro, we turned to investigate the pathway’s phenotypic roles in the cellular regulation of metabolites associated with nematode mutualism and insect pathogenesis. Clarke and co-workers identified the citric acid cycle as being a critical metabolic hub to support nematode mutualism and secondary metabolism, including anthraquinone and stilbene pathways, in P. luminescens (43). Derepression of hexA is also known to upregulate anthraquinone and stilbene polyketides (44), the former implicated in anti-predation of the bacteria-nematode complex (45) and the latter required for antibiosis and nematode development (46). In the citric acid cycle study, the Clarke group used transposon screens to identify malate dehydrogenase (mdh), a NAD+-dependent enzyme that converts malate to oxaloacetate (and reducing equivalent NADH) within the citric acid cycle (43). While the authors noted the major overall importance of the citric acid cycle and its role in generating reducing equivalents for cellular growth and secondary metabolite synthesis, they also argued that other citric acid cycle-associated regulatory pathways were likely at play. Our prior proteomic/metabolomic data and current biochemical data suggest that the pepteridine genetic locus could be one such pathway. As noted above, our previous quantitative proteomic data supported that the pepteridine genetic locus positively affects pyrone autoinducer and biosynthetic pathway enzymes in a ∆hexA background. Consequently, we proposed that the pathway could participate in signaling associated with the mutualist/pathogen transition (23). Additionally, our C-domain biochemical data here further support the upstream utilization of α-ketobutyrate (in the formation of 1) and citric acid ACS Paragon Plus Environment

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cycle substrates, pyruvate (2) and potentially oxaloacetate (3), in the biosynthesis of the pepteridines. To assess this connection between the citric acid cycle and the pepteridine pathway in the production of metabolites associated with nematode mutualism and secondary metabolism, we analyzed the relative stilbene and anthraquinone production levels between an established ∆hexA strain (25) and our pepteridine genetic locus mutant constructed in this strain, ∆hexA/locus. Indeed, deletion of the pepteridine genetic locus significantly reduced stilbene (46) and stilbene epoxide (47) production levels relative to the controls, further supporting a positive regulatory role for the pepteridine genetic locus (Figures 4 and S23). For the known anthraquinones (48), the extent of SAM-dependent methylation was affected. In the locus mutant, the doubly methylated anthraquinones were significantly reduced whereas the mono-methylated analogs were elevated or remained the same (Figures 4 and S24). Collectively, these data support regulatory connections among the citric acid cycle, the pepteridine pathway, the LysR regulator hexA involved in the mutualist/pathogen transition, and secondary metabolite synthesis associated with nematode mutualism and antibiosis (Figure 5).

Figure 4. Comparison of Major Stilbene and Anthraquinone P. luminescens Metabolites. The mean integration values from extracted ion chromatograms of stilbene, stilbene epoxide, and anthraquinones are presented. Error bars represent the standard deviation. Statistical significance is denoted as: ns, P>0.05; **, P≤0.01; and ****, P≤0.0001 under the conditions of our experiments.

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Figure 5. Proposed Regulatory Interactions of the Pepteridine Genomic Island. Bold-faced text and blue arrows are metabolic interactions associated with this study. In conclusion, we provide biochemical evidence that the Plu2796 C domain is able to condense short acyl-units (acetyl-, propionly-, and malonyl-) onto a free, non-functionalized tetrahydropterin core, “blocking” the redox active pterin substrate and leading to the tertiary cis-amide products known as the pepteridines. Synthesis similarly leads to cis-amide products, indicating that while the enzyme is likely regioselective, such selectivity is not required to access the observed products. The biochemical and metabolomic data presented herein, in conjunction with our prior genetic, metabolomic, proteomic, and isotopic labeling studies (23), further support that the pepteridine locus contributes to the upregulation of quorum sensing and secondary metabolic enzymes. This is achieved through the utilization of αketoacid metabolites that support the citric acid cycle, rather than free acyl-CoAs, in conjunction with the free tetrahydropterin substrate. Our in vitro biochemical and cellular biological assays indicate that N5-acylation of 5,6,7,8-tetrahydropterin mediated by the C domain may contribute to signaling while sequestering the redox active tetrahydropterin activity. While our collective data support a functional linkage among the citric acid cycle, the pepteridine genetic locus, pyrone quorum sensing, and secondary metabolism associated with nematode mutualism and antibiosis, future experiments are needed to establish the specific biological and regulatory roles of the novel pteridine-NRPS-derived hybrid metabolites at the molecular level. Acknowledgements. Our work on the characterization of specialized metabolites is supported by the Searle Scholars Program (13-SSP-210), the Damon Runyon Cancer Research Foundation (DRR-39-16), the Burroughs Wellcome Foundation (ID #1016720), the Camille and Henry Dreyfus Foundation (TC-17-011), the National Institutes of Health (1DP2-CA186575), and Yale University.

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[19] Challinor, V. L., and Bode, H. B. (2015) Bioactive natural products from novel microbial sources Ann. N. Y. Acad. Sci. 1354, 82-97 DOI: 10.1111/nyas.12954 [20] Farmer, J. J., 3rd, Jorgensen, J. H., Grimont, P. A., Akhurst, R. J., Poinar, G. O., Jr., Ageron, E., Pierce, G. V., Smith, J. A., Carter, G. P., Wilson, K. L., and et al. (1989) Xenorhabdus luminescens (DNA hybridization group 5) from human clinical specimens J. Clin. Microbiol. 27, 1594-1600 [21] Gerrard, J., Waterfield, N., Vohra, R., and ffrench-Constant, R. (2004) Human infection with Photorhabdus asymbiotica: an emerging bacterial pathogen Microb. Infect. 6, 229-237 DOI: 10.1016/j.micinf.2003.10.018 [22] Costa, S. C., Girard, P. A., Brehelin, M., and Zumbihl, R. (2009) The emerging human pathogen Photorhabdus asymbiotica is a facultative intracellular bacterium and induces apoptosis of macrophage-like cells Infect. Immun. 77, 1022-1030 DOI: 10.1128/IAI.01064-08 [23] Park, H. B., Perez, C. E., Barber, K. W., Rinehart, J., and Crawford, J. M. (2017) Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster Elife 6, e25229 DOI: 10.7554/eLife.25229 [24] Joyce, S. A., and Clarke, D. J. (2003) A hexA homologue from Photorhabdus regulates pathogenicity, symbiosis and phenotypic variation Mol. Microbiol. 47, 1445-1457 [25] Langer, A., Moldovan, A., Harmath, C., Joyce, S. A., Clarke, D. J., and Heermann, R. (2017) HexA is a versatile regulator involved in the control of phenotypic heterogeneity of Photorhabdus luminescens PLoS One 12, e0176535 DOI: 10.1371/journal.pone.0176535 [26] Craig, J. W., and Brady, S. F. (2011) Discovery of a metagenome-derived enzyme that produces branched-chain acyl-(acyl-carrier-protein)s from branched-chain alpha-keto acids ChemBioChem 12, 1849-1853 DOI: 10.1002/cbic.201100215 [27] Brachmann, A. O., Reimer, D., Lorenzen, W., Augusto Alonso, E., Kopp, Y., Piel, J., and Bode, H. B. (2012) Reciprocal cross talk between fatty acid and antibiotic biosynthesis in a nematode symbiont Angew. Chem. Int. Ed. Engl. 51, 12086-12089 DOI: 10.1002/anie.201205384 [28] Peng, C., Pu, J. Y., Song, L. Q., Jian, X. H., Tang, M. C., and Tang, G. L. (2012) Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line Proc. Natl. Acad. Sci. U.S.A. 109, 8540-8545 DOI: 10.1073/pnas.1204232109 [29] Fu, C., Auerbach, D., Li, Y., Scheid, U., Luxenburger, E., Garcia, R., Irschik, H., and Muller, R. (2017) Solving the puzzle of one-carbon loss in ripostatin biosynthesis Angew. Chem. Int. Ed. Engl. 56, 2192-2197 DOI: 10.1002/anie.201609950 [30] Groehn, V., Frohlich, L., Schmidt, H., and Pfleiderer, W. (2000) Pteridines, Part CXI, Pteridinebased photoaffinity probes for nitric oxide synthase and aromatic amino acid hydroxylases Helv. Chim. Acta. 83, 2738-2750 DOI: 10.1002/1522-2675(20001004)83:103.0.co;2-a [31] Rehse, J., and Pfleiderer, W. (2009) Pterdines CXX. Synthesis and properties of tetrahydropterins coupled to 1,4-dihydropyridines Heterocycles 77, 953-970 DOI: 10.3987/com-08-s(f)70 [32] Wei, C. C., Crane, B. R., and Stuehr, D. J. (2003) Tetrahydrobiopterin radical enzymology Chem. Rev. 103, 2365-2383 DOI: 10.1021/cr0204350 [33] Davydov, R., Ledbetter-Rogers, A., Martasek, P., Larukhin, M., Sono, M., Dawson, J. H., Masters, B. S., and Hoffman, B. M. (2002) EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound L-arginine or N(G)hydroxyarginine Biochemistry 41, 10375-10381 [34] Bobst, A. (1967) Über Pterinchemie 23. Mitteilung [1]. Radikalbildung während der Oxydation von Tetrahydrofolsäure und 5,6,7,8-Tetrahydropterin Helv. Chim. Acta 50, 2222-2225 DOI: 10.1002/hlca.19670500807 [35] Ehrenberg, A., Hemmerich, P., Muller, F., and Pfleiderer, W. (1970) Electron spin resonance of pteridine radicals and the structure of hydropteridines Eur. J. Biochem. 16, 584-591 DOI: 10.1111/j.1432-1033.1970.tb01121.x [36] Nagatsu, T., Mizutani, K., Nagatsu, I., Matsuura, S., and Sugimoto, T. (1972) Pteridines as cofactor or inhibitor of tyrosine hydroxylase Biochem. Pharmacol. 21, 1945-1953 DOI: 10.1016/00062952(72)90007-X

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[37] Kaufman, S. (1967) Pteridine cofactors Annu. Rev. Biochem. 36, 171-184 DOI: 10.1146/annurev.bi.36.070167.001131 [38] Murr, C., Widner, B., Wirleitner, B., and Fuchs, D. (2002) Neopterin as a marker for immune system activation Curr. Drug. Metab. 3, 175-187 DOI: 10.2174/1389200024605082 [39] Lane, A. N., and Fan, T. W. (2015) Regulation of mammalian nucleotide metabolism and biosynthesis Nucleic Acids Res. 43, 2466-2485 DOI: 10.1093/nar/gkv047 [40] Gomtsyan, A., Didomenico, S., Lee, C. H., Stewart, A. O., Bhagwat, S. S., Kowaluk, E. A., and Jarvis, M. F. (2004) Synthesis and biological evaluation of pteridine and pyrazolopyrimidine based adenosine kinase inhibitors Bioorg. Med. Chem. Lett. 14, 4165-4168 DOI: 10.1016/j.bmcl.2004.06.029 [41] Bowers, S., Truong, A. P., Ye, M., Aubele, D. L., Sealy, J. M., Neitz, R. J., Hom, R. K., Chan, W., Dappen, M. S., Galemmo, R. A., Jr., Konradi, A. W., Sham, H. L., Zhu, Y. L., Beroza, P., Tonn, G., Zhang, H., Hoffman, J., Motter, R., Fauss, D., Tanaka, P., Bova, M. P., Ren, Z., Tam, D., Ruslim, L., Baker, J., Pandya, D., Diep, L., Fitzgerald, K., Artis, D. R., Anderson, J. P., and Bergeron, M. (2013) Design and synthesis of highly selective, orally active Polo-like kinase-2 (Plk-2) inhibitors Bioorg. Med. Chem. Lett. 23, 2743-2749 DOI: 10.1016/j.bmcl.2013.02.065 [42] Zhou, W., Liu, X., Tu, Z., Zhang, L., Ku, X., Bai, F., Zhao, Z., Xu, Y., Ding, K., and Li, H. (2013) Discovery of pteridin-7(8H)-one-based irreversible inhibitors targeting the epidermal growth factor receptor (EGFR) kinase T790M/L858R mutant J. Med. Chem. 56, 7821-7837 DOI: 10.1021/jm401045n [43] Lango, L., and Clarke, D. J. (2010) A metabolic switch is involved in lifestyle decisions in Photorhabdus luminescens Mol. Microbiol. 77, 1394-1405 DOI: 10.1111/j.13652958.2010.07300.x [44] Kontnik, R., Crawford, J. M., and Clardy, J. (2010) Exploiting a global regulator for small molecule discovery in Photorhabdus luminescens ACS Chem. Biol. 5, 659-665 DOI: 10.1021/cb100117k [45] Joyce, S. A., Lango, L., and Clarke, D. J. (2011) The Regulation of Secondary Metabolism and Mutualism in the Insect Pathogenic Bacterium Photorhabdus luminescens Adv. Appl. Microbiol. 76, 1-25 DOI: 10.1016/B978-0-12-387048-3.00001-5 [46] Joyce, S. A., Brachmann, A. O., Glazer, I., Lango, L., Schwar, G., Clarke, D. J., and Bode, H. B. (2008) Bacterial biosynthesis of a multipotent stilbene Angew. Chem. Int. Ed. Engl. 47, 19421945 DOI: 10.1002/anie.200705148 [47] Park, H. B., Sampathkumar, P., Perez, C. E., Lee, J. H., Tran, J., Bonanno, J. B., Hallem, E. A., Almo, S. C., and Crawford, J. M. (2017) Stilbene epoxidation and detoxification in a Photorhabdus luminescens-nematode symbiosis J. Biol. Chem. 292, 6680-6694 DOI: 10.1074/jbc.M116.762542 [48] Brachmann, A. O., Joyce, S. A., Jenke-Kodama, H., Schwar, G., Clarke, D. J., and Bode, H. B. (2007) A type II polyketide synthase is responsible for anthraquinone biosynthesis in Photorhabdus luminescens ChemBioChem 8, 1721-1728 DOI: 10.1002/cbic.200700300

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Figure 1. The Pepteridine Biosynthetic Gene Cluster and Key NRPS-Pteridine Coupling Reaction. The biosynthetic gene cluster responsible for the formation of pepteridine A (1) and B (2) is shown. Green genetic regions denote pteridine synthesis machinery, blue genetic regions denote pyruvate dehydrogenaselike machinery, and red genetic regions highlight NRPS-like regions. The predicted function of each gene is noted – T, thiolation; and C, condensation. The inset represents the predicted biosynthesis in which free tetrahydropterin is functionalized with an acyl appendage present on the phosphopantetheinyl arm of the T domain through the action of the C domain. The known products of the pathway, pepteridine A (1) and B (2) are shown (metabolite color codes correspond to associated genetic regions in the pathway).

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Figure 2. In vitro Biochemical Assessment of Plu2796-C Activities. Extracted ion chromatograms within 10 ppm of the calculated exact monoisotopic protonated mass for each molecule are shown for each experimental condition. 1–3 have been confirmed by co-elution studies using synthetic standards.

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Scheme 1. Synthesis of Pepteridine A (1) and B (2). Reagents and conditions: (a) isobutyric anhydride, pyridine, reflux, 5 h, 76.9%; (b) PtO2, H2, rt, 24 h, 71.5%; (c) propionyl chloride for 4c, rt, 10 min, 54.5%; acetyl chloride for 4d, rt, 10 min, 53.0%; (d) MeOH, CH3ONa, rt, 4 h, 79.1% for 1 and 79.7% for 2. See supporting information for synthesis of enzymatic product 3.

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Figure 3. Radical Scavenging Activities. The ability of pepteridine A (1), pepteridine B (2), and 5,6,7,8tetrahydropterin to quench the stable free radical di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) indicator is shown. l-Ascorbic acid and tetrahydropterin exhibit half-maximal inhibitory concentrations (IC50) of 26 and 23 µM, respectively.

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Figure 4. Comparison of Major Stilbene and Anthraquinone P. luminescens Metabolites. The mean integration values from extracted ion chromatograms of stilbene, stilbene epoxide, and anthraquinones are presented. Error bars represent the standard deviation. Statistical significance is denoted as: ns, P>0.05; **, P≤0.01; and ****, P≤0.0001 under the conditions of our experiments.

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Figure 5. Proposed Regulatory Interactions of the Pepteridine Genomic Island. Bold-faced text and blue arrows are metabolic interactions associated with this study.

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