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A Radical Approach to Enzymatic #-Thioether Bond Formation Alessio Caruso, Leah B. Bushin, Kenzie A. Clark, Ryan J. Martinie, and Mohammad R. Seyedsayamdost J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b11060 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Journal of the American Chemical Society

A Radical Approach to Enzymatic β-Thioether Bond Formation Alessio Caruso,† Leah B. Bushin,† Kenzie A. Clark,† Ryan J. Martinie,† and Mohammad R. Seyedsayamdost†,‡,* †

Department of Chemistry, Princeton University, Princeton, NJ 08544, United States Department of Molecular Biology, Princeton University, Princeton, NJ 08544, United States KEYWORDS Radical SAM Enzymes, Natural Products, Streptococci, RiPPs

‡

ABSTRACT: Ribosomally-synthesized and post-translationally-modified peptides (RiPPs) are an emerging class of natural products that harbor diverse chemical functionalities, usually introduced via the action of a small number of tailoring enzymes. We have been interested in RiPP biosynthetic gene clusters that encode unusual metalloenzymes, as these may install yet unknown alterations. Using a new bioinformatic search strategy, we recently identified an array of unexplored RiPP gene clusters that are quorum sensing-regulated and contain one or more uncharacterized radical S-adenosylmethionine (RaS) metalloenzymes. Herein, we investigate the reaction of one of these RaS enzymes and find that it installs an intramolecular β-thioether bond onto its substrate peptide by connecting a Cys-thiol group to the β-carbon of an upstream Asn residue. The enzyme responsible, NxxcB, accepts several amino acids in place of Asn and introduces unnatural β-thioether linkages at unactivated positions. This new transformation adds to the growing list of Nature’s peptide macrocyclization strategies and expands the already impressive catalytic repertoire of the RaS enzyme superfamily.

INTRODUCTION Ribosomally-synthesized and post-translationally-modified peptides (RiPPs) have recently emerged as a structurally and functionally diverse family of natural products with often selective biological activities.1-4 The production of RiPPs relies on a unique biosynthetic logic: A precursor peptide is produced ribosomally from the canonical twenty amino acids. It contains a core region, which receives posttranslational modifications by one or more tailoring enzymes, as well as leader and, in some cases, follower sequences, which are removed during the maturation process to deliver the final natural product. RiPPs characterized to date boast numerous unusual structural motifs.1-4 Despite the variety of modifications, however, their biosynthesis typically requires few proteins and is therefore particularly suited to bacteria that do not possess large genomes, such as host-associated bacteria in mammalian microbiomes. We previously elucidated the structure and biosynthesis of streptide, a 9-mer RiPP natural product from one such commensal bacterium, Streptococcus thermophilus.5 We were especially intrigued by the regulation and structure of streptide: its production is induced at high cell densities by a shp/rgg quorum sensing (QS) system that is conserved in streptococci.6-8 Structurally, streptide features a unique carboncarbon bond between the sidechains of Lys and Trp, which is manufactured by the radical S-adenosylmethionine (RaS) enzyme StrB. RaS enzymes form one of the largest and most diverse superfamilies in Nature, comprising more than 330,000 annotated members to date.9-12 They bind a [4Fe-4S] cluster, usually via a conserved CxxxCxxC motif, and catalyze the reductive cleavage of S-adenosylmethionine (SAM) to give a 5'-deoxyadenosyl radical (5'-dA•), which abstracts a hydrogen atom from the substrate to initiate a diverse array of chemical outcomes. StrB is a member of the SPASM-domain

containing RaS enzymes, named after founding members involved in the maturation of subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin.13-17 The SPASM domain is a C-terminal extension that facilitates binding of one or more four-iron-four-sulfur [4Fe-4S] clusters, thus enhancing the catalytic capabilities of RaS enzymes. Motivated by the novelty of the StrB-catalyzed Lys-Trp crosslink during streptide biosynthesis, we recently conducted a bioinformatic search by coupling the signatures of the RaS enzyme superfamily, encoded in the vicinity of a short precursor peptide, to an upstream shp/rgg QS operon, reasoning that the corresponding RiPPs are likely produced at high cell densities and may contain novel scaffolds. This bioinformatic search yielded ~600 unexplored RaS enzyme-associated RiPP gene clusters, which we refer to as RaS-RiPPs.18 When arranged into a sequence similarity network (SSN) based on precursor peptide sequences, we observed 16 distinct subfamilies of RaS-RiPPs (Fig. 1A). Gratifyingly, the str, aga, and sui clusters, which encode RaS enzymes that install Lys-Trp crosslinks onto their respective substrates, located to a single subfamily,5,19-22 suggesting that distinct chemical reactions may be catalyzed by RaS enzymes in each of the remaining 15 subfamilies. Indeed, our investigations of the WGK cluster showed that the corresponding RaS enzyme, WgkB, catalyzes a four-electron oxidation in an unprecedented Trp modification resulting in the formation of a tricyclic tetrahydrobenzindole motif (Fig. 1A). These results forecast the possible chemical novelty contained within the RaS-RiPPs network. In the present study, we examine the reaction catalyzed by the NxxC subfamily and find that the corresponding RaS enzyme, NxxcB, installs a β-thioether crosslink onto its substrate peptide by linking a Cys-thiol group with the unactivated βmethylene position of an upstream Asn residue, the first such reaction for the RaS enzyme family. β-thioether bonds are a

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well-known feature of lantipeptides, where they are introduced by a heterolytic mechanism involving dehydration of a Ser or Thr residue followed by conjugate addition of a nearby Cys sidechain.23,24 By contrast, NxxcB builds β-thioether crosslinks via a radical mechanism, thereby expanding the modification to any β-carbon-containing amino acid and providing an alternative pathway by which macrocycles can be introduced during RiPP biogenesis.

broad 395 nm transitions, symptomatic of [4Fe-4S]2+ clusters (Fig. 2B). The presence of [2Fe-2S] could be ruled out based on UV-visible absorption spectra of the as-isolated and reconstituted protein (Fig. S1). Quantitation of Fe and S2- gave 4.9 ± 0.4 Fe and 5.7 ± 0.5 S2- for the as-isolated protein and 10.7 ± 0.6 Fe and 10.7 ± 0.4 S2- per protein monomer after reconstitution. The X-band EPR spectrum of reduced NxxcB revealed an apparently axial signal. Spectral deconvolution showed that the observed signal was a composite consisting of a major component (85-90% intensity) with g = [2.040, 1.905, 1.900] – indicative of one or more [4Fe-4S]+ clusters routinely found in RaS enzymes – and a minor component (10-15% intensity) with g = [2.034, 2.022, 2.001] – consistent with a [3Fe-4S]0 cluster. EPR spin quantitation gave 8 µM spin associated with [4Fe-4S]+ and [3Fe-4S]0, suggesting the majority of clusters were EPR-silent. In conjunction with the bioinformatic prediction of a SPASM domain within NxxcB (Fig. S2-S3), and cleavage of SAM in the absence of substrate to generate 5'-dA (Fig. S4), our data are in line with the presence of a [4Fe-4S]+ cluster, presumably in the active site, and one or two auxiliary [4Fe-4S]2+ clusters, with minor levels of a [3Fe-4S]0 replacing one of these clusters in a subpopulation of the enzyme. This conclusion may be assessed further by additional biophysical methods in the future.25,26

Figure 1. RaS-RiPPs in Streptococci. (A) SSN of Ras-RiPPs based on precursor peptide sequences. The subfamilies are named using conserved motifs in each peptide.18 Reactions catalyzed by StrB and WgkB (red bonds) are shown. The NxxC subfamily is highlighted in red. (B, C) S. orisratti (B) and S. porci (C) NxxC gene cluster. Genes coding for the precursor peptides, RaS enzymes, transporters, hypothetical genes, and QS operons (consisting of shp and rgg) are rendered in black, red, blue, white, and green, respectively. The sequences of both precursor peptides are shown.

RESULTS AND DISCUSSION The NxxC cluster and characterization of NxxcB. The NxxC gene cluster is shown (Fig. 1B, C). It is regulated by an upstream shp/rgg QS operon and encodes a precursor peptide (NxxcA), a RaS enzyme (NxxcB), two transporter/proteases (NxxcC/D), and a hypothetical protein (NxxcE). We focused on two NxxC clusters from Streptococcus orisratti (Fig. 1B) and Streptococcus porci (Fig. 1C), commensal bacteria found in rat and pig oral microbiomes, respectively. Given the precedents of StrB, AgaB, SuiB, and WgkB,5,18,20 we hypothesized that NxxcB would modify the precursor peptide first, and that the other genes would contribute to processing and exporting the peptide. We therefore began by assessing the reactivity of NxxcB with NxxcA. NxxcB from S. orisratti was expressed heterologously in Escherichia coli (Tables S1-S2), purified under anaerobic conditions (Fig. 2A), and chemically reconstituted with Fe and S2-.22,25 Analysis of reconstituted NxxcB by UV-visible absorption spectroscopy revealed the characteristic 315 nm and

Figure 2. Characterization of NxxcB from S. orisratti. (A) SDSPAGE gel of purified NxxcB. (B) UV-vis spectrum of purified and reconstituted NxxcB (16 µM). (C) X-band EPR spectrum of reconstituted, reduced NxxcB at 10 K (50 μW, 9.37 GHz). (D) EPR spectral simulation of the trace in panel (C) yields a major signal (85-90% of total intensity) with g = [2.040, 1.905, 1.900] and a minor signal (10-15% total intensity) with g = [2.034, 2.022, 2.001]. The traces are color-coded as indicated.

Reaction catalyzed by NxxcB. Reconstituted NxxcB was next reacted with synthetic NxxcA and SAM under anoxic conditions. Initial analysis by HPLC-Qtof-MS revealed the presence of a product peak with a -2 Da mass relative to substrate. However, control samples lacking NxxcB also contained the -2 Da product, presumably due to intramolecular disulfide bond formation during the aerobic workup. To preclude this reaction, three Cys mutants were synthesized: C23S-, C28S-, and C23S/C28S-NxxcA (Fig. 1B). Overnight reactions of each substrate with NxxcB and SAM resulted in


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Journal of the American Chemical Society nearly complete conversion to a -2 Da product with C28SNxxcA, small amount of product with C23S-NxxcA, and no product at all with the double mutant (Figs. 3A, S5-S6; Table S3). Product formation with the single mutants was only observed in the presence of NxxcB and SAM. Kinetic analysis gave a Vmax/[ET] of 4.8 ± 1.5 min-1 with C28S-NxxcA. Moreover, experiments with iodoacetamide revealed labeling with C28S-NxxcA, but not after this substrate had been reacted with NxxcB (Table S3). Together, these data suggest that NxxcB catalyzes a modification involving C23. Further evidence for this conclusion came from highresolution tandem MS (HR-MS/MS) analysis: the C28SNxxcA product was treated with trypsin and the fragments subjected to HR-MS/MS, which showed collision-induced dissociation at each peptide bond in the tryptic fragment, except for those within the N20–C23 sequence. Fragment b ions upstream of N20 and y ions downstream of C23 were -2 Da relative to substrate, suggesting macrocyclization connecting these residues. (Fig. 3B, Tables S4-S5). A similar result was obtained with the orthologous S. porci NxxcB, which was expressed and purified in an identical manner. Reaction with the S. porci wt NxxcA also yielded a product peptide with a mass -2 Da relative to substrate and a fragmentation pattern indicative of crosslink formation between the analogous residues N19 and C22 (Fig. 3C, Table S6). These observations are consistent with a NxxcB-catalyzed modification that links the nearby Cys and Asn residues in each precursor peptide.

Figure 3. Reaction of NxxcB with NxxcA. (A) Extracted ion intensity of -2 Da products of S. orisratti NxxcB with C23S/C28S-NxxcA (green trace), C23S-NxxcA (red), and C28SNxxcA (blue). (B) HR-MS/MS analysis of trypsin-treated C28SNxxcA after reaction with NxxcB from S. orisratti. No fragments were observed between N20 and C23. (C) HR-MS/MS analysis of wt NxxcA after reaction with NxxcB from S. porci. No fragments were observed between N19 and C22.

Structural elucidation of the NxxcB product. Complete structural elucidation was next conducted using 1D/2D NMR. The S. orisratti C28S-NxxcA/B reaction was carried out on a large scale and the HPLC-purified product subjected to extensive NMR analysis. The spectra were compared to those of the unmodified substrate, which allowed us to completely assign the product NMR spectra and to focus on the unique correlations that report on the post-translational modification (Figs. S7-S9, Table S7). Overlaid HSQC and gCOSY spectra are

shown as well as a product NOESY spectrum (Fig. 4A–C). Most importantly, the N20-H was significantly downshifted (2.88 to 3.86 ppm) accompanied by a conversion from a methylene in the substrate (39.0 ppm) to a methine in the product (54.5 ppm) as identified by HSQC analysis, thus pinpointing the -carbon of N20 as the site of the modification (Fig. 4A). The N20-H was detectable but was also deshielded in the product (from 4.53/54.9 ppm to 5.25/53.5 ppm). These NMR data are strongly suggestive of a C-S bond linking N20 and C23 (Fig. 4B, D). Consistent with this conclusion were HMBC data and NOE correlations between the N20-H and the C23H (Fig. 4C, D), a cross-peak not observed in the substrate NOESY spectrum. Moreover, the 1H and 13C shifts for the intervening Asp and Ser residues were altered in the product. In particular, the HSQC Hα–Cα correlation of D21 was distinct in the substrate (4.64/51.3 ppm) and product (4.65/52.5 ppm). The S22-Hα shift also changed subtly upon thioether bond formation (4.40 to 4.49 ppm), as did the shifts of S22-Hβ/Cβ (3.82/61.2 ppm in the substrate vs. 3.90/59.2 ppm in the product). Together, these data provide compelling evidence for a β-thioether bond be-

tween N20 and C23 installed by NxxcB, the first such reaction demonstrated to date for a RaS enzyme (Fig. 4E, F).

Figure 4. NMR analysis of the NxxcB product. (A) Overlaid DEPT-edited HSQC spectra of the unmodified C28S-NxxcA (green CH/CH3, blue: CH2) and the converted product (red: CH/CH3, blue: CH2). Key correlations are marked. (B) Overlaid gCOSY spectra of the unmodified C28S-NxxcA (green) and the converted product (red). The arrow points to the N20-H–H correlation. (C) NOESY spectrum of the cyclized product. The arrows point to the N20-H–H and the N20-H–C23-H correlations. (D, E) Relevant NMR correlations (D) used to solve the structure of the NxxcB-catalyzed crosslink (E). The β-carbon stereochemistry at N20 in the product remains to be determined. (F) Reaction catalyzed by NxxcB.



Mechanistic investigations. Several mechanisms can be envisioned for β-thioether crosslink formation. Akin to the pathway proposed by Marahiel and colleagues for -thioether bonds, one of the auxiliary Fe-S clusters could activate the Cys residue for reaction with the Asn C-radical, generated by 5ꞌ-dA•, to deliver the product concomitant with reduction of the auxiliary cluster (Fig. 5A).27,28 Alternatively, abstraction of the H or H of N20 would yield a radical intermediate with a mildly acidic adjacent 1H. Deprotonation and oxidation would yield an ,-unsaturated di-amide, a good electrophile for conjugate addition by a Cys-thiolate (Fig. 5B).29 Interestingly, the - and -carbons are both electrophilic in the putative Michael acceptor in the second mechanism (Fig. 5B), making positioning of the Cys-thiolate critical for regiospecific thioetherification. We assessed three aspects of these pathways: ligation of C23 to an auxiliary cluster in the first mechanism (Fig. 5A), wash-in of a solvent proton into the product unique to the second (Fig. 5B), and recycling of reducing equivalents, possibly relevant to both. We tested the first feature via the approach of Flühe et al, who showed that, like a redox change from [4Fe-4S]2+ to [4Fe4S]+, ligation of a Cys-thiolate to an Fe-S cluster suppresses the extinction coefficient leading to lowered absorption at 400 nm.27 Indeed, incubation of NxxcB with wt NxxcA resulted in decreased absorption at 315 nm and 395 nm, the features associated with [4Fe-4S]2+ (Fig. 5C). A similar shift is observed upon reduction of the [4Fe-4S]2+ with Na2S2O4. In contrast, incubation of NxxcB with C23S/C28S-NxxcA did not lead to significant spectral changes (Fig. 5D). Reaction of each single mutant, C23S-NxxcA and C28S-NxxcA, also resulted in decreased absorption (Fig. S10). While these experiments do not provide conclusive proof, they are consistent with ligation of a Cys residue on NxxcA to one of the [4Fe-4S]2+ clusters in NxxcB, and possibly explain the small amount of turnover observed with C23S-NxxcA. We assessed the second aspect, wash-in of a solvent proton into the product, by conducting enzymatic assays in D2O. All components were exchanged into D2O and the reaction monitored by HPLC-Qtof-MS. HR-MS analysis did not reveal incorporation of a deuteron into the product (Fig. S11). Instead HR-MS and HR-MS/MS data were consistent with a crosslink bearing a 1H at the α-carbon of N20 (Fig. 5E, Tables S8-S9). Although it remains possible that a non-exchangeable -1H is first removed from N20 and then returned to the same position,30 we currently favor the mechanism in Fig. 5A, which provides a working model for future studies. Finally, we explored electron recycling by conducting experiments with pre-reduced NxxcB in the absence of any reductant. Reaction of pre-reduced NxxcB (~8 μM spin) with NxxcA and SAM gave ~240 µM product, indicating 30 turnovers in the absence of an external reductant. This result is entirely consistent with recycling of reducing equivalents during the catalytic cycle. A similar phenomenon has previously been observed with other SPASM-domain containing RaS enzymes.20,31 NxxcB substrate tolerance. β-Thioether connections are a hallmark of lantipeptides. Given that NxxcB installs these via a disparate mechanism relative to lantipeptide cyclases, we wondered whether acceptor residues other than Asn would serve as a substrate for the enzyme. Four variants were synthesized: N20Q-, N20D-, N20E-, and N20A-NxxcA. All peptides also contained the C28S substitution to avoid intramolecular disulfide bond formation. Each substrate was reacted with

Figure 5. Mechanism of -thioetherification by NxxcB. (A, B) Radical (A) vs. heterolytic (B) pathways for β-thioether bond formation; see text for details. (C, D) UV-vis spectral changes upon binding of wt NxxcA (C) or C23S/C28S-NxxcA (D) to NxxcB. Shown are spectra of NxxcB alone (red traces), NxxcB and NxxcA variant (blue traces), and NxxcB after reduction with Na2S2O4 (dotted trace). (E) Reaction of wt NxxcA with NxxcB in D2O gives rise to only protonated product, in line with the mechanism in panel A or a non-exchangeable deprotonation/reprotonation route in panel B.

NxxcB and product formation assessed by HR-MS. After overnight incubation, which results in near-complete product formation with C28S-NxxcA, we observed yields ranging from 7–55% for the four substrate variants (Fig. 6, Table S10). Poor conversion was only observed with N20E-NxxcA. N20A-NxxcA was readily accepted by the enzyme giving rise to a lanthionine crosslink. The Vmax/[ET] determined mirrored the conversion yields (Table S10). These results indicate


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Journal of the American Chemical Society that NxxcB can accept alternative substrates and install nonnatural β-thioether bonds.

Figure 6. Reaction of NxxcB with substrate analogs containing Gln, Glu, Asp, or Ala at residue 20. Product formation was observed with all four variants, with the conversion yields shown relative to C28S-NxxcA. See Table S10.

CONCLUSIONS RaS enzymes have previously been shown to install αthioether linkages during sactipeptide biosynthesis via a homolytic mechanism.27,28 β-Thioether bonds, however, are a characteristic feature of lantipeptides. In this case, the C-S bond is installed by a heterolytic pathway in which the sidechains of Ser or Thr are dehydrated in an ATP or Glu-tRNAdependent fashion to generate the Michael acceptor. Conjugate addition of the Cys-thiolate then completes the reaction.32,33 As a result of this mechanism, the acceptor amino acids in lanthionines are limited to Ser or Thr. We herein report the first βthioether crosslink generated by the RaS enzyme NxxcB. Because RaS enzymes rely on radical chemistry, the acceptor residue may in theory be any of the 20 canonical amino acids, sans Gly, thus extending the scope of residues that may undergo this type of modification. Our results with several substrate variants, notably N20Q- and N20A-NxxcA, are in line with this notion. The reaction catalyzed by NxxcB expands the already impressive repertoire of reactions known to be catalyzed by RaS enzymes; it also highlights that additional new modifications remain to be discovered from the abundance of RaS-RiPPs gene clusters in streptococci and other bacteria. MATERIALS AND METHODS Materials and strains. All materials were purchased from Sigma-Aldrich or Fisher Scientific unless otherwise specified. Streptococcus orisratti DSM 15617 and Streptococcus porci DSM 23759 were obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures). Restriction enzymes, T4 DNA ligase, Q5 High-Fidelity DNA Polymerase, Shrimp Alkaline Phosphatase, and Trypsin-Ultra (Mass Spectrometry Grade) were purchased from New England BioLabs (NEB). DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT). All Fmoc- and side chain-protected amino acids, low-loading Fmoc-Gly-Wang resin and other components for solid-phase peptide synthesis were purchased from EMD Millipore except for HATU and HOAt, which were obtained from GenScript. General procedures. UV-visible spectra were acquired on a Cary 60 UV-visible spectrophotometer (Agilent). HPLC separations were carried out on an Agilent 1260 Infinity Series analytical or preparative HPLC system equipped with a temperaturecontrolled column compartment, a photodiode array detector and an automated fraction collector. The analytical system was also equipped with an automated liquid sampler. Low resolution HPLC-MS analysis was performed on an Agilent instrument con-

sisting of a liquid autosampler, a 1260 Infinity Series HPLC system coupled to a photodiode array detector and a 6120 Series ESI mass spectrometer. Samples were resolved on a reversed-phase Phenomenex Luna C18 column (3 μm, 4.6 x 100 mm). Both MeCN and H2O contained 0.1% (v/v) formic acid. Highresolution (HR) HPLC-MS and HR-tandem HPLC-MS were carried out on an Agilent 6540 Accurate-Mass Q-tof LC-MS system, consisting of a 1260 Infinity Series HPLC, an automated liquid sampler, a photodiode array detector, a JetStream ESI or Dual ESI source, and the 6540 Series Q-tof. Samples were separated on a reversed-phase Phenomenex Luna C18 column (3 μm, 4.6 x 100 mm) or an Agilent C18 Eclipse (5 μm, 150 x 4.6 mm) using water and MeCN as (+0.1% formic acid) as mobile phase. Elution was carried out isocratically with 5% MeCN in water (10 min) followed by a gradient of 5–44% MeCN over 17 minutes, and finally 44–95% MeCN over 5 minutes, at a flow rate of 0.5 mL/min. NMR and EPR spectra were acquired at the Princeton University Department of Chemistry Facilities. NMR spectra were collected in D2O or H2O/D2O (9:1), in the triple resonance cryoprobe of a Bruker A1 Avance III 500 MHz NMR spectrometer. 1D/2D NMR data were analyzed with MestReNova software. CW X-band EPR spectra were recorded at 10 K on Bruker EMXplus EPR spectrometer with an HS1059 X-band high sensitivity cavity and an Oxford liquid Helium cryostat. Cloning of NxxcB. Genomic DNA from both S. orisratti DSM 15617 and S. porci DSM 23759 was isolated using the Wizard Genomic DNA Purification Kit (Promega). The S. orisratti nxxcB gene (IMG Gene ID: 2515615110) was PCR-amplified from S. orisratti genomic DNA using Q5 DNA Polymerase (NEB) in FailSafe Buffer D (Epicenter) and primers nxxcB_DSM15617_C and nxxcB_DSM15617_D (SacI) (Table S2). A second fragment containing a 5ꞌ-His tag was generated using primers nxxcB_DSM15617_A (NcoI) and nxxcB_DSM15617_B (Table S2) and fused to the nxxcB gene fragment by overlap extension PCR. The S. porci nxxcB gene (IMG Gene ID: 2523721627) was PCR-amplified from S. porci genomic DNA using Q5 DNA Polymerase (NEB) in FailSafe Buffer D (Epicenter) and primers nxxcB_DSM23759_F (NdeI) and nxxcB_R (HindIII) (Table S2). PCR products were purified using the Qiagen PCR Purification Kit and subsequently digested with the restriction enzymes NcoI and SacI (S. orisratti) or NdeI and HindIII (S. porci). Vector pET28b(+) (Novagen) was digested with the corresponding restriction enzymes (NcoI/SacI or NdeI/HindIII) and subsequently treated with Shrimp Alkaline Phosphatase (NEB). Following gel extraction using the Qiagen Gel Extraction Kit, inserts and vectors were ligated using T4 DNA Ligase (NEB). The ligation reaction was transformed into E. coli DH5α cells by heat shock. Single colonies were screened for insertion of the fragment. Sequence verified pET-28b(+)_nxxcB constructs for both orthologs (S. orisratti and S. porci) along with pDB1282 were co-transformed by heat shock into the expression host E. coli BL21(DE3). Expression, purification, and reconstitution of NxxcB. Expression, purification, and reconstitution of NxxcB was carried out using previously published procedures without modifications.22 Purified NxxcB originating from both S. orisratti and S. porci was quantified using the method reported by Barr et al.34 NxxcB enzymes was anaerobically reconstituted with 10-fold excess FeII and 10-fold excess Na2S, as reported.22 EPR spectroscopy. 300 μL of ~100 μM His-NxxcB was incubated with 10 mM Na2S2O4 for 30 minutes and then transferred to an EPR tube. The EPR tube was capped, removed from the glovebox, and immediately frozen in liquid N2. The following parameters were used for spectral acquisition: microwave frequency, 9.3717 GHz; microwave power, 50 μW; modulation amplitude, 0.8 mT; time constant, 5 ms. To quantify the concentration of free spin, spectra of prereduced NxxcB (see below) were recorded and compared to a



CuSO4 standard, prepared as previously described.25 The sample and standard were measured using the following, identical conditions: microwave frequency, 9.3802 GHz; microwave power, 20 μW; modulation amplitude, 1 mT; time constant, 20 ms. Comparison of the doubly integrated intensities indicated a spin concentration in the pre-reduced sample of 8 ± 2 μM. Simulation of EPR spectra. EPR spectra were analyzed using the "pepper" utility from the EasySpin software package.35 The spectrum of NxxcB was simulated using two primary spectral components. The first component was simulated with g = [2.040, 1.905, 1.900] and ~90% of the spectral intensity, consistent with the presence of one or more [4Fe4S]1+ cluster(s).36 The second component was simulated with g = [2.034, 2.022, 2.001] and ~10% of the spectral intensity, consistent with the presence of some [3Fe4S]0 clusters, which we attribute to degradation of the [4Fe4S]1+ cluster(s) by contaminating oxygen during sample preparation or to incomplete reconstitution.36 Synthesis of NxxcA variants by SPPS. NxxcA substrates were prepared by Fmoc-based solid phase peptide synthesis (SPPS) on a preloaded Fmoc-Gly-Wang-resin using a Liberty Blue automated peptide synthesizer equipped with a Discover microwave module (CEM). The deprotection solution consisted of 10% piperazine (w/v) in a 10:90 solution of EtOH:NMP (N-methylpyrrolidine) supplemented with 0.1 M HOBt (1-hydroxybenzotriazole). The activator solution consisted of 0.5 M DIC (N,N'-diisopropylcarbodiimide) in DMF and activator base solution of 1.0 M Oxyma with 0.1 M DIPEA in DMF. A typical coupling cycle used 5 equiv. of amino acid and 5 equiv. of coupling reagent was used. Residues 1–4, 23, 27 and 28 were double-coupled. The second coupling of residue 2 was performed using 0.45 M HATU as activator instead of DIC and 2 M DIPEA as base instead of 1.0 M Oxyma. The synthesis was typically performed on a 100 μmol scale. Upon completion of the synthesis, the resin was removed from the reaction vessel and transferred to an Econo-Pac column (BioRad). The resin was washed several times with DMF, followed by DCM, and dried thoroughly under vacuum. The peptide was cleaved from the resin by incubation with freshly prepared cleavage cocktail (5 mL per 100 mg resin) consisting of 90% TFA, 2.5% H2O, 2.5% TIS (triisopropylsilane), and 5% βME (2mercaptoethanol). The reaction was stirred for 3 h at room temperature. The mixture was drained from the reaction tube and the resin was rinsed several times with TFA. The filtrate and rinses were combined and subsequently concentrated by evaporation of TFA under a stream of N2. The peptide was then precipitated by addition of 10 volumes of ice-cold diethyl ether and isolated by centrifugation (4000g, 10 min, 4°C). The ether was poured off and the peptide was dried overnight in a fume hood. Purification of NxxcA and variants. Crude NxxcA was dissolved in 15% MeCN (+0.1% formic acid) and purified by preparative HPLC. Manual injections were performed on a preparative Phenomenex Jupiter C18 column (7 μm, 2.5 x 25 cm), which was equilibrated in 10% MeCN in H2O (+0.1% formic acid). The peptide was eluted with a gradient of 5–40% MeCN over 17 minutes. The purity of NxxcA was verified by HPLC-Qtof-MS and NMR analysis (Table S3 and Fig. S7). Enzymatic activity assays. Enzymatic assays were performed in an inert atmosphere within an MBraun glovebox. The NxxcA substrate peptides were transported into the glovebox as lyophilized material and resuspended in water supplemented with 2.5 mM dithiothreitol (DTT) to give a ~10 mM solution. Sodium dithionite (DT), DTT, and SAM were also transported into the glovebox as powders and prepared as stock solutions in water. Reactions were typically carried out in 0.5 mL microcentrifuge tubes on a 30 μL scale with final concentrations of 2.5 mM DTT, 2.5 mM DT, 0.25–0.30 mM substrate peptide, 25 μM NxxcB, and 0.8 mM SAM. Incubation times ranged from 30 sec to 10 h depending on the experiment. At the end of the incubation period,

the reaction tubes were taken out of the glovebox and immediately quenched by incubation at 95 °C for 3 minutes. Precipitated protein was removed by centrifugation (21,000g, 5 min). Samples were diluted twofold with water (+0.1% formic acid) prior to injection onto the HPLC-Qtof-MS. Enzymatic reactions in D2O. Enzymatic assays in D2O were carried out in the same manner as those described above except that stock solutions of all components (C28S-NxxcA, DTT, DT, and SAM) were prepared in D2O instead of H2O. In addition, NxxcB was exchanged into a D2O based buffer (50 mM Tris, 20 mM KCl, 5% glycerol, pD 7.0) using a Micro Bio-Spin 6 column (Bio-Rad) according to manufacturer’s instructions. Enzymatic reactions with pre-reduced NxxcB. To assess the possibility of electron recycling in the reaction of NxxcB, ~400 μL of 100 μM NxxcB was incubated with 10 mM DT for 30 minutes. The reductant was then removed by size exclusion chromatography and NxxcB was split into two batches – one for EPR spin quantitation, and the other for enzymatic assays. Enzymatic assays were carried out in duplicate in 0.5 mL microcentrifuge tubes on a 30 μL scale with final concentrations of 0.25 mM C28S-NxxcA, 25 μM NxxcB, and 0.8 mM SAM. Large-scale reaction. In order to generate sufficient material for NMR analysis, a large-scale reaction (7.5 mL) with C28SNxxcA as substrate was carried out. C28S-NxxcA (7 mg) was transported into the glovebox as a lyophilized powder in a 15 mL falcon tube, to which the other reaction components were directly added. The concentrations of all components were identical to those in the small-scale assays described above. The reaction was incubated at room temperature for 48 h, then brought out of the glovebox and heat-quenched at 95 °C for 3 minutes. Product formation was confirmed by HPLC-Qtof-MS. Isolation of converted C28S-NxxcA product from largescale reaction. The C28S-NxxcA product was purified from the reaction mixture by HPLC using a Phenomenex Synergi FusionRP column (4 μm, 250 x 10 mm). Samples were injected automatically and resolved with a gradient of 5–30% MeCN over 12 minutes at a flow rate of 2.5 mL/min. Fractions containing the peptide were pooled and lyophilized. The lyophilized material was resuspended in 0.5 mL H2O (+0.1% formic acid) and purified further using a Grace Vydac C18 column (5 μm, 250 x 4.6 mm). Elution was carried out with a gradient of 5–17.5% MeCN over 25 minutes, followed by a gradient of 17.5–100% over 10 min at a flow rate of 1.0 mL/min. Product eluted at 24–25 min (~13% MeCN) and the substrate at 26 min (~14% MeCN). Fractions containing product were pooled and lyophilized. Structural elucidation of NxxcB product. 1H, gCOSY, TOCSY, DEPT-edited gHSQC, gHMBC, and NOESY NMR spectra were analyzed to elucidate the structure of the product of the reaction of C28S-NxxcA with NxxcB (Table S7 and Fig. S7S9). Spectral features for residues not involved in the macrocycle did not change relative to the those for the NxxcA substrate. The shifts of residues that are involved in the modification, on the other hand, displayed significant changes.

ASSOCIATED CONTENT Supporting Information HR-MS and HR-MS/MS analysis of substrates and products of the reaction with NxxcB, 1D/2D NMR spectra and spectral annotation for the substrate and product peptides, analysis of the reaction of NxxcAB in D2O, and of pre-reduced NxxcB with substrate and SAM. This material is available free of charge via the Internet at: http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author


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Journal of the American Chemical Society *[email protected]

Funding Sources This work was supported by the Burroughs Wellcome Fund PATH Investigator Award (to M.R.S.). L.B.B. was supported by a National Science Foundation Graduate Research Fellowship.

Notes The authors declare no competing financial interest.

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