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Mechanistic Investigations of Lysine-Tryptophan Crosslink Formation Catalyzed by Streptococcal Radical SAM Enzymes Kelsey R Schramma, Clarissa C. Forneris, Alessio Caruso, and Mohammad R. Seyedsayamdost Biochemistry, Just Accepted Manuscript • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Biochemistry

Mechanistic Investigations of Lysine-Tryptophan Crosslink Formation Catalyzed by Streptococcal Radical SAM Enzymes Kelsey R. Schramma†, Clarissa C. Forneris†, Alessio Caruso†, 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

*Correspondence: [email protected]

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ABSTRACT Streptide is a ribosomally synthesized and post-translationally modified peptide with a unique cyclization motif consisting of an intramolecular lysine-tryptophan crosslink. Three radical SAM enzymes, StrB, AgaB, and SuiB from different Streptococci, have been shown to install this modification onto their respective precursor peptides in a leader-dependent fashion. Herein we conduct detailed investigations to differentiate between several plausible mechanistic proposals, specifically addressing radical versus electrophilic addition to the indole during crosslink formation, the role of substrate side-chains in binding in the enzyme active site, and the identity of the catalytic base in the reaction cycle. Our results are consistent with a radical electrophilic aromatic substitution mechanism for the key carbon-carbon bond-forming step. They also elaborate on other mechanistic features that underpin this unique and synthetically challenging post-translational modification.

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Biochemistry

INTRODUCTION Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging family of natural products with complex structures and intriguing biological activities.1-3 Among natural products, the biosynthesis of RiPPs follows a unique logic and can be contrasted with that of non-ribosomal peptides (NRPs).4 While NRPs are built one amino acid at a time from large biosynthetic enzymes, and therefore correspondingly large biosynthetic gene clusters, RiPP biogenesis commences with the ribosomal production of a multi-partite precursor peptide, consisting of a core sequence as well as N-terminal (leader) and/or C-terminal (follower) sequences. The peptide is subsequently modified at the core sequence, usually by a small number of tailoring enzymes, upon which the leader and/or follower sequences are removed to deliver the mature product. With this simple strategy, RiPPs can achieve structural complexity rivaling that of NRPs, but with a much smaller genomic footprint. Key to the complexity of RiPPs are tailoring enzymes that carry out new or unusual transformations. We recently discovered one such enzyme, StrB, that cyclizes its corresponding precursor peptide, StrA, by installing an unprecedented intramolecular carbon-carbon crosslink between the β-methylene group of a Lys residue and the C7-indole of a Trp residue (Figure 1).5 We further showed that pathogenic Streptococci express similar enzymes, specifically AgaB and SuiB form Streptococcus agalactiae and Streptococcus suis, which install analogous crosslinks in their respective precursor peptides, AgaA and SuiA.6 StrB, AgaB, and SuiB are members of the radical S-adenosylmethionine (SAM) enzyme superfamily, which characteristically employ a reduced [4Fe-4S]+ cluster to homolytically cleave SAM, thus generating a 5ꞌ-deoxyadenosyl radical (5ꞌ-dA•) and L-Met.7-10 The 5ꞌ-dA• initiates catalysis, usually by abstraction of an unactivated H-atom to generate a substrate radical, with the subsequent steps specific to a given enzyme/transformation. Reductive cleavage of SAM is structurally executed by a partial triose phosphate isomerase (TIM) barrel that houses the SAMbinding [4Fe-4S]+ cluster.11 Aside from the TIM barrel, StrB, AgaB, and SuiB also contain additional, conserved protein domains important for Lys-Trp crosslink formation, which we recently visualized by solving X-ray crystal structures of SuiB in apo-form as well as with bound SAM or SAM and SuiA.6,11-14 N-terminal to the partial TIM barrel is a so-called RiPP recognition element (RRE) domain that has been proposed to recognize the leader sequence within the precursor peptide and thus provide specificity.15 C-terminal to the TIM barrel is a so-called SPASM domain, an extension that allows binding of two additional ‘auxiliary’ [4Fe-4S] clusters,

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Aux-I and Aux-II.11-14 A key Cys ligand is provided by the bridging domain, which sits between the partial TIM barrel and the SPASM motif, and makes key contacts to SuiA.14 The mechanistic and structural studies conducted thus far, have provided important clues regarding the possible functions of these various protein domains and the mechanism of LysTrp cyclization.5 Nonetheless, questions remain about the nuanced roles of the RRE during the catalytic cycle and the detailed chemistry underlying this transformation. Specifically, the key carbon-carbon bond-forming step during Lys-Trp cyclization may proceed by an electrophilic aromatic substitution (EAS) reaction, with Trp synthase,16,17 Trp halogenases,18,19 and Trp prenyltransferases20 providing exemplars of this mechanistic paradigm in Biology. Alternatively, a radical variation on this theme, radical EAS, may be operative as we have suggested before.5 Herein, we experimentally distinguish between these possibilities, and expand on our previous studies on AgaB and SuiB by addressing the site of initial H-atom abstraction, by modulating key precursor peptide binding determinants, and by identifying a candidate Brønsted base during the catalytic cycle. Our results rule out EAS and instead provide further evidence for a radical EAS mechanism.5 The new insights allow us to elaborate a mechanistic model for this challenging and unusual transformation, catalyzed by a new subfamily of RiPP-modifying radical SAM enzymes. A

strA

strB

strC

B

9mer

MSKELEKVLESSSMAKGDGWKVMAKGDG

StrB StrC

Streptide

Figure 1. Structure and biosynthesis of streptide. The str cluster encodes a precursor peptide (strA) with the sequence shown, a radical SAM enzyme (strB), and a protease/transporter (strC). The core sequence of StrA is shown in bold and crosslinked residues in red. AgaB and SuiB catalyze a similar reaction on a conserved K-GDG-W motif in their respective precursor peptides, AgaA and SuiA. (B) X-ray crystal structure of SuiB.14 Domains are colored as follows: N-terminal RRE (red), radical SAM domain (green); bridging domain (blue); SPASM domain (purple). The active site FeS cluster in the radical SAM domain, Aux-I, and the peripheral Aux-II cluster are all shown in grey ball-stick models.

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Biochemistry

MATERIALS AND METHODS Materials and strains. The genomic DNA of Streptococcus suis 92-4172 was kindly provided Prof. Marcelo Gottschalk at the University of Montreal.21 Streptococcus agalactiae ATCC 13813 was obtained from the ATCC. Bacterial growth media were purchased from Becton Dickinson. Kanamycin, ampicillin, (NH4)2Fe(SO4)2, arabinose, IPTG, PMSF, lysozyme, βME, DTT, Na2S2O4, SAM, DMPD, zinc acetate, FeCl3, ferrozine, DIPEA, TIS, TFA, CuSO4, BOC anhydride, Fmocsuccinimide, NaHCO3, 8-hydroxyquinoline, AgOTf, 2,6-lutidine, and formic acid (FA) were purchased from Sigma-Aldrich. 2,6,6-2H3-L-lysine was purchased from CDN isotopes. DNase I and Talon metal affinity resin were from Clontech. Proof-reading Q5 DNA polymerase, restriction enzymes, T4 DNA ligase, and chemically-competent E. coli cells were purchased from New England Biolabs. All PCR reactions were performed in Failsafe Buffer D (Epicentre). All Fmocand side-chain-protected natural amino acids, Fmoc-homo-Ser(Trt)-OH, low-loading FmocLys(BOC)-Wang and Fmoc-Val-Wang resins were purchased from EMD Millipore. FmocOrn(BOC)-OH, Fmoc-Dab(BOC)-OH, Fmoc-Dap(BOC)-OH, Fmoc-Hnl-OH, (S)-Fmoc-2-amino5-tritylsulfanyl-pentanoic acid, and Fmoc-homo-Cys(Trt)-OH were obtained from Chem-Impex. HATU and HOAt were from GenScript. DNA purification kits were purchased from Qiagen. Synthesis of H-α,ε,ε-2H3-L-Lys(BOC)-OH. Nε-BOC-protected α,ε,ε-2H3-Lys was synthesized using reported methods with minor modifications.22 To a 25 mL pear-shaped flask equipped with a stir bar were added 2 mL of 1 M NaHCO3 and 125 mg of α,ε,ε-2H3-L-Lys (0.66 mmol, 1 eq.). The amino acid was dissolved by stirring for several minutes at RT, then CuSO4 (0.35 mmol, 0.53 eq.) was dissolved in 1 mL H2O and combined with the Lys solution, which was supplemented with 1.5 mL of 1 M NaHCO3. 210 mg of BOC-anhydride (0.965 mmol, 1.4 eq.) was dissolved in 0.75 mL acetone and added dropwise to the mixture. The reaction was stirred for 24 h, then 1 mL of MeOH was added and stirring was continued for another 18 h. The mixture was filtered through a fine-fritted funnel and the filter washed with 5 mL of 1 M NaHCO3, 1 mL H2O, and 1 mL of MeOH. The precipitated Lys-Cu complex was scratched off the filter, transferred to a 50 mL round-bottom flask, resuspended in 11.5 mL H2O, and supplemented with 114.3 mg of solid 8-hydroxyquinoline (0.787 mmol, 1.1 eq.). The mixture was stirred overnight, filtered, and the filtrate extracted with 3 x 30 mL of EtOAc. The organic layers were combined and washed with 5 mL H2O. The aqueous layer was combined with the H2O washes, frozen, and lyophilized yielding 83 mg (325 μmol) of H-α,ε,ε-2H3-L-Lysine(BOC)-OH in 50% yield.

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Synthesis of Fmoc-α,ε,ε-2H3-L-Lys(BOC)-OH. Fmoc-protection was carried out as previously reported.23 Briefly, 83 mg of α,ε,ε-2H3-L-Lysine(BOC)-OH (325 μmol) was dissolved in 3 mL of water, supplemented with 34 mg of Na2CO3 (325 μmol , 1 eq.), and the solution chilled in an ice bath. 156 mg Fmoc-succinimide (455 μmol, 1.4 eq.) was dissolved in 3 mL of MeCN and added dropwise to the solution. The reaction was stirred overnight at room temperature, then diluted with 12 mL water. Unreacted Fmoc-succinimide was extracted with diethyl ether (2 x 10 mL). The pH of the combined aqueous layers was adjusted to 2–3 with 2 M HCl and the precipitated product then extracted into EtOAc (3 x 10 mL). The combined organic extracts were rinsed with saturated NaCl, dried over Na2SO4, filtered, and concentrated in vacuo to yield 107 mg of the desired product (70%), which was used without further purification. The identity of the product was confirmed by HPLC-MS and NMR. [M+H]+calc = 372.2 (BOC-group is removed due to insource fragmentation), [M+H]+obs = 372.2. 1H NMR (500 MHz, DMSO): δ 7.81 (d, J = 7.44, 2H), 7.66 (d, J = 7.44, 2H), 7.57 (s, 1H), 7.34 (t, J = 7.44, 2H), 7.26 (t, J = 7.40, 2H), 6.71 (s, 1H), 4.22 (d, J = 6.94, 2H), 4.16 (t, J = 6.37, 1H), 1.45-1.7 (m, 4H), 1.29 (s, 9H) 1.20-1.27 (m, 2H). Synthesis of Fmoc-Hnl(Trt)-OH. To a mixture of AgOTf (0.58 g, 2.25 mmol, 3 eq.) and 2,6lutidine (262 μL, 2.25 mmol, 3 eq) in 2.5 mL DCM was added a solution of TrtCl (0.625 g, 2.25 mmol, 3 eq.) dissolved in 2 mL DCM at 0°C. The resulting yellow suspension was supplemented with Fmoc-Hnl-OH (554.1 mg, 0.75 mmol, 1 eq) and 2 mL DCM. The reaction was stirred for 1 hr at 0 °C, then warmed to RT and left to stir overnight. After addition of 500 μL MeOH, the reaction mixture was stirred for 15 min and the solvents removed in vacuo. Fmoc-Hnl(Trt)-OH was purified by silica gel chromatograph (hexane/EtOAc, 1:1). 1H NMR (500 MHz, CCl3) δ = 7.77 (t, J = 6.16, 2H), 7.60 (d, J = 7.54, 2H), 7.45 (d, J = 7.75, 6H), 7.40 (t, J = 7.43, 2H), 7.277.34 (m, 9H), 7.24 (t, J = 7.43, 2H), 5.20 (d, J = 8.23, H), 4.44 (m, J = 7.26, 2H), 4.39 (t, J = 7.26, H), 4.23 (t, J = 7.99, H), 3.09 (t, J = 6.05, 2H), 1.68 (m, 4H), 1.48 (m, 2H). Synthesis and purification of AgaA and SuiA analogs. All peptides were prepared using SPPS as previously described.6 Unnatural amino acids were incorporated using 2 eq. of the Fmoc-protected form, 1.9 eq. HATU, 1.9 eq. HOAt, and 4 eq. DIPEA. The coupling reaction was allowed to proceed for 90 min. Cleavage of the full-length peptide from resin proceeded as described to give crude AgaA and SuiA,6 which were purified by HPLC. Crude AgaA or SuiA were dissolved in 10% MeCN (+ 0.1% FA) and purified by repeated injections onto a Phenomenex Jupiter C18 column (7 μm, 2.5 x 25 cm), which was equilibrated in 10% MeCN in H2O (+ 0.1% FA). The peptide was eluted with a gradient of 10-40% MeCN over 22 minutes.

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Biochemistry

When necessary, the material was reapplied to the same column with the same elution program to give pure peptide, as verified by HR-HPLC-MS and NMR analysis (Table S1, Figure S1). Generation of E319A-suiB and E318A-agaB. The E319A-SuiB mutant was generated by first creating two fragments (A and B) each with the desired mutation. The fragments were then fused by overlap extension PCR and cloned into pET-28b(+). Fragment A, containing the NdeI cut site, was generated with primers suiB_E319A_B and suiB_clone_A on the template plasmid pET-28b(+)_suiB (Table S2). Fragment B was generated using pET-28b(+)_suiB as a template with primers suiB_E319A_A and suiB_ clone_B, which contained the BamHI cut site (Table S2). Fragments A and B were isolated using a Qiagen PCR purification kit and then joined using overlap extension PCR and primers suiB_clone_A and suiB_clone_B. The resulting full-length mutant gene was purified, digested with NdeI and BamHI, visualized on an agarose gel, purified using a Qiagen gel extraction kit, and ligated into pET-28b(+) to give vector pET28b(+)_suiB(E319A). The mutant was confirmed by sequencing the entire gene. The E318AAgaB mutant was generated in an analogous manner using primers agaB_E319A_A, agaB_ E319A_B, agaB_clone_A, and agaB_clone_B to give plasmid pET-28b(+)_agaB(E318A). E319A-SuiB and E318A-AgaB were expressed and purified as previously described.5,6 Wt enzymes and the Fe-S cluster mutants were available from a previous study.6 Enzymatic Activity Assays. Activity assays were generally performed in a 50 μL reaction containing buffer (100 mM HEPES, 300 mM KCl, 10% glycerol, 5 mM DTT, pH 7.5), AgaB or SuiB (10–25 μM), Na2S2O4 (2 mM), and substrate (1 mM). At t=0, 25 μL were removed and reacted with 25 μL 100 mM H2SO4 to provide the no-reaction control. For the remaining 25 μL, the reaction was initiated by addition of 1 mM SAM, and allowed to proceed for 1 hour (enzyme mutants, deuterated substrates) or 20 hours (Lys analogs) before quenching with 25 μL of 100 mM H2SO4. The precipitated protein was removed by centrifugation at 14,000g for 3 min. The supernatant was applied to a Phenomenex Strata C8 SPE column (50 mg), which had been equilibrated with H2O + 0.1% FA. The column was washed with 2 mL H2O and peptides eluted with 1.5 mL of 50% MeCN in H2O and 0.5 mL 100% MeCN. The eluates were combined, dried in vacuo, and analyzed by HPLC-Qtof-MS (see SI Methods).

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RESULTS AND DISCUSSION Mechanistic proposals for AgaB/SuiB. Previous studies with StrB have provided two key insights regarding the mechanism of Lys-Trp crosslink formation.5,6 First, we demonstrated abstraction of an H-atom from the β-methylene group of a Lys side-chain by 5ꞌ-dA•, without a kinetic isotope effect on Vmax. Second, we showed that the auxiliary clusters were important in turnover. Removal of Aux-I rendered a largely unstable protein, while removal of the second cluster, AuxII, abolished turnover. With these criteria in mind, at least two mechanisms may be proposed for Lys-Trp crosslink formation. In the first – the radical addition mechanism (Figure 2A) – the lysyl radical adds into the π system of the indole ring to form the Lys-Trp bond and a tryptophanyl radical. This step, addition of an alkyl radical to an aromatic framework, has plentiful precedent in synthetic organic chemistry and has been proposed in enzymatic reactions as well.24-28 Rearomatization via oxidation by a Fe-S cluster, formally consisting of proton and electron transfer reactions, then completes the modification. A second mechanism, predominantly based on Trp synthase and FAD-dependent Trp chlorinases,16-19 is also consistent with the available data. In this canonical EAS mechanism (Figure 2B), the lysyl radical is oxidized via deprotonation of the now acidic α-1H and electron transfer to an auxiliary cluster to generate an α-β unsaturated amide. Michael addition of the indole to the acceptor, followed by reprotonation completes the crosslink. In both mechanisms, resetting the redox states of the Fe-S clusters allows for multiple turnovers.

Figure 2. Mechanistic proposals for Lys-Trp crosslink formation. (A) Radical addition mechanism. (B) Electrophilic aromatic substitution mechanism. See text for details.

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Biochemistry

By studying the requirements for Lys-Trp crosslink formation, we recently observed that the Lys ε-amine is essential.6 No product was observed in an AgaA variant, where the Lys residue was replaced with norleucine. These data led us to suggest that the side-chain amine may act as a catalytic base or provide an important anchoring point in the enzyme active site. To distinguish between these mechanisms (Figure 2), we have conducted experiments with α-2HLys, a variety of Lys derivatives to replace the Lys2 ε-amine with more or less basic groups, and appropriate side-directed mutants of AgaB and SuiB. Reaction of α-2H-Lys2-AgaA/SuiA with AgaB/SuiB. The key difference between the radical addition and EAS mechanisms, is that the α-H at Lys2 is lost in the latter. We therefore set out to distinguish between these by incorporating α-2H-Lys into the precursor peptides. Commercially available α,ε,ε-2H3-Lys was protected with BOC (ε-amine) and Fmoc (α-amine) groups, and the isotopomer then inserted at Lys2 of AgaA and SuiA by solid-phase peptide synthesis (SPPS). Subsequently, α,ε,ε-2H3-Lys2-AgaA ([M+2H]+2 = 1172.08918) was reacted with AgaB and the products interrogated using HPLC-Qtof-MS. The mechanisms in Figure 2A and 2B predict formation of a product that is 2 Da or 3 Da lighter than the substrate, respectively. HR-MS analysis of the crosslinked product yielded [M+2H]+2 of 1171.08135 (Figure 3A), consistent with a loss of two protons. A -3 Da product was not observed when the reaction was quenched at various time points. Moreover, assessment of 5ꞌ-dA after the reaction showed that it was exclusively protonated (Figure 3B). Lastly, with Vmax/[E]T values of ~0.18 min-1 for AgaA and α,ε,ε-2H3-Lys2-AgaA, no isotope effect was observed on turnover. Analogous results were obtained with SuiB and α,ε,ε-2H3-Lys2-SuiA. The α-2H was not lost during the course of the reaction and only protonated 5ꞌ-dA was formed (Figure 3B). Combined with our previous studies,5 which showed that only one H-atom is abstracted from the side-chain of Lys2, these results preclude formation of a carbocation or α,β-unsaturated amide as an intermediate, thereby ruling out an EAS mechanism (Figure 2B). Role of auxiliary Fe-S clusters in turnover. In StrB, the Aux-II [4Fe-4S] cluster is required for Lys-Trp crosslink formation.5 The recent X-ray crystal structure of SuiB shows that Aux-I is held by four Cys residues, Cys321, Cys347, Cys406, and Cys419.14 Interestingly, while the latter three are located within the SPASM domain, Cys321 is provided by the so-called bridging domain, and this cluster therefore serves as a linchpin to hold together the SPASM and bridging domains adjacent to the active site in the modified TIM barrel (Figure 1B). Aux-I is ~16 Å away from the active site cluster that cleaves SAM. Aux-II, on the other hand, is ligated by the

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Biochemistry

remaining four Cys residues in the SPASM domain, including Cys409 and Cys415; it is ~10 Å away from Aux-I and ~27 Å from the active site Fe-S cluster. We generated two double mutants in each enzyme to test the role of the active site and Aux-II Fe-S clusters: C121A/C124A-SuiB and the analogous C120A/C123A-AgaB to verify the role of the active site cluster, as well as C409A/C415A-SuiB and C408A/414A-AgaB, which abolished binding of Aux-II as determined by EPR spectroscopy and quantification of Fe and labile S2-,6 to test the role of Aux-II. Soluble enzyme proved difficult to obtain with the Aux-I mutant, perhaps underlining the important structural role of this cluster. Activity assays with wt and mutant AgaB or SuiB showed that C120A/C123A-AgaB, C408A/414A-AgaB, and C121A/C124A-SuiB were entirely inactive in turnover (Figure 4A). With SuiB, however, the Aux-II mutant, C409A/C415A-SuiB, was able to support turnover at a rate ~60% relative to wt SuiB (Figure 4B). This latter result is consistent with an involvement of Aux-I as the oxidant of the tryptophanyl radical intermediate, a conclusion that is corroborated by the distance constrains inferred from the crystal structure of wt SuiB. At this point, we cannot explain the different enzymatic activities of the Aux-II mutants in SuiB and AgaB, but believe that Aux-I carries out the same redox function in both enzymes.

A

B

MS Intensity (counts)

*

1171.0

1173.0

*

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1151.5

m/z

1153.5

Figure 3. Reaction of AgaB/SuiB with α,ε,ε-2H3-Lys2-AgaA/SuiA. (A) Schematic of the observed product in the AgaAB reaction: Substrate (m/z)calc = 1171.09230; product (m/z)calc = 1171.08448. The m/z values shown correspond to the [M+2H]+2 ion. Note that the [M-Met+2H]+2 m/z values are shown, as we routinely observed loss of the N-terminal Met residue in our assays, possibly due to a contaminating protease. (B) Mass spectrum of the product of the reaction of α,ε,ε-2H3-Lys2-AgaA with AgaB (top) and α,ε,ε-2H3-Lys2-SuiA with SuiB. The m/z values for the AgaB reaction are shown in panel (A). Following values were obtained for the SuiB reaction: Substrate (m/z)calc = 1152.59078; Substrate (m/z)obs = 1152.59977; product (m/z)calc = 1151.58295, product (m/z)obs = 1151.59366.

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Biochemistry

A

B

Figure 4. Requirement of Fe-S clusters for enzymatic activity in AgaB and SuiB. (A) Extracted ion chromatogram for the product ion in the reaction of wt AgaA with wt AgaB (blue trace), wt AgaA with the Aux-II mutant C408A/C414A-AgaB (red trace), and wt AgaA with the active site Fe-S cluster mutant C120A/C123A-AgaB (black trace). (B) Extracted ion chromatogram for the product ion in the reaction of wt SuiA with wt SuiB (blue trace), wt SuiA with the Aux-II mutant C409A/C415A-SuiB (red trace), and wt SuiA with the active site Fe-S cluster mutant C121A/C124A-SuiB (black trace).

Role of Lys2-AgaA in turnover. We next attempted to clarify the role of the Lys2 ε-amine, which, as mentioned above, is essential for turnover. The strict requirement for this moiety leads us to postulate two possible roles: (1) anchoring the substrate in the active site for proper positioning via H-bonding or ion-pair interactions, or (2) serving as a Brønsted base in the course of the catalytic cycle (Figure 5A). In the first scenario, decreasing the length of the Lys side-chain, while maintaining the amino group, would be expected to diminish turnover. Conversely, maintaining the side-chain length but replacing the amine with substituents capable of H-bonding interactions with higher (OH) or lower (SH) pKa values, should not affect turnover. In the second scenario, where Lys2 acts as an intramolecular base, a 7-membered ring transition state would be formed during the deprotonation reaction of the tryptophanyl radical. Decreased Lys side-chain length would lead a 6-membered or 5-membered ring, and given the relative stability of these, a significant effect on catalysis would not be expected. On the other hand, replacement of the ε-amine with a ε-hydroxyl group would be expected to reduce or abolish reactivity. Thus, the outcome of experiments in which the length of the Lys2 side-chain is contracted and the ε-amine is replaced with hydroxyl or thiol groups, would allow distinction between the two proposed roles for Lys2 (Figure 5A). We obtained Lys analogs that were commercially available or accessible by simple chemical synthetic schemes. Shown in Figure

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5B, these consist of ornithine (Orn), diaminobutyric acid (Dab) and diaminopropionic acid (Dap), a side chain contraction from four methylene units (Lys) to one (Dap). We also synthesized and incorporated 6-hydroxynorleucine (Hnl), homoserine (Hser), 5-thio-2-aminopentanoic acid (Pcys) and homocysteine (Hcys), residues containing 4, 3, or 2 side-chain methylene groups and a terminal OH or SH. These were reacted with AgaB in four independent replicates and the extent of turnover quantified by HPLC-Qtof-MS, with the averages shown (Figure 5B, 5C). Except for Dap, reactions with all analogs under substrate-saturating conditions resulted in product formation to differing degrees. All products were characterized by HR-MS/MS to verify that the crosslink observed occurred between Lys2 and Trp6 (Table S3, Figure S2). The product of K2Orn-AgaA was further verified by NMR analysis (Figure S3). While product was observed with Hser, the amounts were too little for analysis by HR-MS/MS. Nonetheless, the HR-MS data and the dependence of product formation on SAM and AgaB are all consistent with Lys-Trp crosslink formation in this variant (Table S1). With the NH2-bearing Lys analogs, a direct relationship was observed between the length of the side-chain and the extent of product formation, as Lys gave the highest activity (normalized to 100%), while progressively diminished turnover occurred with Orn (37  19% turnover), Dab (13  8%), and Dap (no turnover, Figure 5B). As rationalized above, these results are consistent with an anchoring role during catalysis. This conclusion was further corroborated with the hydroxyl- and thiol-substituted derivatives. Both Pcys and Hcys resulted in product formation at 10  5% and 2  1% relative to Lys, with the enhanced basicity of the thiolate group resulting in diminished turnover relative to Orn and Dab. The hydroxylated derivatives were active too, with Hnl exhibiting turnover similar to Lys at 63  14%. The homoserine derivative, though active, was turned over less efficiently (2  1%) relative to Dab (13%), with a similar side-chain length. At this point, we cannot explain the reduced turnover with Hser. Nonetheless, given that AgaB turnover is largely independent of the pKa of the side-chain at Lys2-Aga, or even inversely correlated with the basicity of the side-chain, the data favor an anchoring role, rather than a catalytic base function, during turnover. We propose that the Lys2 side-chain, where the modification occurs, is used as contact point by the enzyme active site to ensure proper positioning of the substrate, perhaps in part explaining the strict regio- and stereo-specificity observed in Lys-Trp crosslink formation. That the Lys derivatives, regardless of the length of the side-chain, react more efficiently with the enzyme than the OH- or SH-derivatives, may point to an ion pair interaction between Lys2 and the enzyme active site.

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Biochemistry

A

B

C Lys Hnl

Orn Dab

Pcys

Hcys Hser

Dap

Figure 5. Probing the role of the Lys2 side-chain in catalysis. (A) Two proposed roles: in pathway a, the Lys ε-amine acts as an intramolecular Brønsted base, while in pathway b, this function is carried out by an active site residue and the Lys2 side-chain instead anchors and thereby properly positions the substrate. (B) Lys substitutes inserted at residue 2 of AgaA. The average turnover observed relative to wt AgaA is shown. The star denotes the pKa for the conjugate acid of the side-chain. (C) Extracted ion chromatogram for the cyclized product of AgaA variants, after reaction with AgaB, containing the Lys substitutions shown in panel (B). The color coding is as follows: wt AgaA (dark green), Lys2Orn-AgaA (green), Lys2Dab-AgaA (light green), Lys2Dap-AgaA (neon green), Lys2HnlAgaA (blue), Lys2Hser-AgaA (light blue), Lys2Pcys-AgaA (red), Lys2Hcys-AgaA (orange). n.p. indicates no product detected. Each trace is labeled for clarity.

Role of E318-AgaB/E319-SuiB in catalysis. The results above indicate that a catalytic Brønsted base is likely provided by the enzyme active site or another residue in the substrate.

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We ruled out the possibility of D4-AgaA performing this function as previous studies with D4AAgaA showed only a 3-fold reduction in turnover, inconsistent with an essential role during catalysis.6 The recent crystal structure of SuiB, in which both SAM and SuiA were captured in the active site, pinpointed other potential catalytic base residues.14 Among several candidates, the structure revealed only one titratable residue, Glu319 (Glu318 in AgaB), within 5 Å of the Trp side-chain, which was modeled into the active site as the C-terminal tail of the substrate was not visualized.14 We created E318A-AgaB and E319A-SuiB substitutions to examine the role of this residue in turnover. Measurement of the kinetic parameters of E318A-AgaB gave a kcat of 0.016 ± 0.005 min-1, an ~8-fold reduction relative to wt (kcat ~0.12 ± 0.03 min-1), without a significant effect on Km (50 ± 15 μM and 77 ± 23 μM for wt and E318A-AgaB, respectively). A similar reduction in turnover was observed in the SuiB system with a kcat of 0.18 ± 0.05 min-1 and 0.026 ± 0.008 min-1 for wt and E319A-SuiB, respectively. Because our assays focus on Vmax and the rate-limiting step is likely associated with resetting the redox state of the Fe-S clusters,5 these results do not directly report on the effect of the Glu-to-Ala mutation on the rate constant for deprotonation. It remains possible that E319 functions as a catalytic base or in the relay of protons out of the active site, but additional studies are necessary to test and distinguish between these possibilities. A detailed mechanism for Lys-Trp crosslink formation. The insights above allow us to propose a more detailed mechanistic model for Lys-Trp crosslink formation (Figure 6A). As before, we propose a radical addition mechanism, in which the substrate binds in the active site, properly positioned by interaction with the Lys2 side-chain, and subsequently reacts with 5ꞌ-dA• to allow for abstraction of the pro-S hydrogen-atom of the β-methylene of Lys2, thus generating a lysyl radical. Electrophilic addition of the lysyl radical to the indole ring gives rise to the LysTrp crosslink and an indolyl radical. Deprotonation of this intermediate by E318 (or E319 in SuiB) via removal of the now acidic indole-H7 results in a radical anion, which rearomatizes by electron transfer to Aux-I. Whether these two latter steps occur as a concerted proton-coupled electron transfer step or as two discrete proton transfer/electron transfer reactions, remains to be established. Finally, reduction of the active site Fe-S cluster and oxidation of Aux-I, possibly by Aux-II (or by the active site cluster), enable additional rounds of turnover. Our model assigns a structural and electron transfer role to Aux-I, while Aux-II appears to predominantly serve a redox function, possibly by interaction with external redox proteins.

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Biochemistry

CONCLUSIONS One of the primary advantages of RiPP biosynthetic gene clusters is that the peptide-substrate for the first modification enzyme is known and that it can be readily synthesized by SPPS, thus facilitating incorporation of suitable mechanistic probes. We have leveraged these advantages and created a number of substrate analogs containing unnatural Lys variants, either deuterated at the α-carbon, containing shortened side-chains and/or hydroxyl/thiol groups in place of the Lys2 ε-amine to interrogate the mechanisms of AgaB and SuiB, in conjunction with site-directed mutants. Analysis of the reactions of these substrate and enzyme variants provides further details regarding Lys-Trp crosslink formation. The modification catalyzed by StrB, AgaB, and SuiB may be compared with other enzymatic indole modification reactions. In the biosynthesis of Trp, tryptophan synthase famously utilizes the nucleophilic nature of indole, along with a fascinating channeling mechanism, to assemble the amino acid in an EAS mechanism (Figure 6B).16,17 In two well-known secondary metabolite modification reactions, Trp halogenations and Trp prenylation, the indole again participates in a polar mechanism with electrophiles.18-20 We therefore considered a similar mechanism for LysTrp cyclization, in which the Trp6 indole would react with an α,β-unsaturated electrophile in a Michael addition reaction. Thioether bonds in lanthipeptides, such as nisin, are formed by a similar mechanism with the Cys thiolate acting as the nucleophile.29,30 With AgaB and SuiB, however, we did not find evidence for a canonical EAS mechanism. Instead, we find that LysTrp crosslink formation occurs, to the best of our knowledge, by a new mechanism involving indole modifications, namely radical electrophilic aromatic substitution (rEAS). In this case the electrophile is not provided by a dehydrated amino acid (Trp synthase),16 hypochlorous acid (Trp halogenase),18 or an allylic carbocation (Trp prenylation),20 but by an electrophilic radical species (Figure 6B, step a). The product of the reaction of this radical with indole is formally a sigma complex, but instead of the carbocationic nature in EAS, it is a radical sigma complex (Figure 6B, step b). The complex then loses a hydrogen atom, rather than a proton as is typical in EAS, to render the final product (Figure 6B, step c). Our previous finding in which the less nucleophilic Trp6Bzt-AgaA (where Bzt is benzothienyl-Ala) yielded cross-linked product is also consistent with a rEAS mechanism, rather than a nucleophilic attack by the indole.6 It is wellknown that radicals react with aromatic groups to give rise to new carbon-carbon bonds. Numerous investigators have provided examples of this phenomenon in radical SAM enzymatic systems.25-28 Within the context of biological indole reactions, however, which predominantly

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proceed by EAS when unsubstituted, rEAS represents a new mechanism. The reaction mediated by PqqE, which was very recently reconstituted in vitro,31 likely provides a further example of an enzyme-catalyzed rEAS. Aside from the enhanced mechanistic understanding, the promiscuity of AgaB observed with the various AgaA analogs suggests that AgaB may be used to chemo-enzymatically synthesize streptide derivatives, which may aid in elucidating the native roles of these cyclic peptides in Streptococci. These studies along with reactions of other radical SAM enzymes in RiPP biosynthetic pathways are currently in progress.

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Biochemistry

A

B

Figure 6. Enzyme-catalyzed rEAS mechanisms. (A) Updated model for Lys-Trp crosslink formation. The purple dome above Lys2 indicates interaction with the enzyme active site via the ε-amine. Reductive cleavage of SAM yields a 5ꞌ-dA• (step a), which abstracts the pro-S H-atom of Lys2, to generate a lysyl radical (step b). Electrophilic addition of the lysyl radical to the indole ring forms a crosslink and an indolyl radical (step c). Deprotonation of the acidic indole-H7 proton, possibly by E319 (SuiB numbering), gives rise to a radical anion (step d), which rearomatizes via oxidation by Aux-I to give product (step e). Dissociation of product (step f) followed by resetting of the redox states of the active site cluster and Aux-I (step g) allow for further turnovers. The active site cluster and Aux-I are shown in red and blue, respectively. Steps d and e may occur by proton-coupled electron transfer, rather than discrete proton transfer/electron transfer steps, as shown. (B) Contrasting EAS (top) and rEAS (bottom) mechanisms for Trp modification. See text for a description.

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ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: General procedures, HR-MS data for substrates and products, HR-MS/MS data for products.

AUTHOR INFORMATION Corresponding author E-mail: [email protected] Telephone: (609) 258-5941 ORCID: Funding We gratefully acknowledge the National Institutes of Health (Grant DP2-AI-124786) and the Burroughs Wellcome Fund (PATH Investigator Award) for funding this work.

ACKNOWLEDGEMENTS We thank Dr. Katherine M. Davis for helpful discussions and Joe Boerma for technical assistance with synthesizing some AgaA variants.

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Biochemistry

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Biochemistry

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