Exploring the Flexibility of the Glycopeptide Antibiotic Crosslinking Cascade for Extended Peptide Backbones

The glycopeptide antibiotics (GPAs) are a clinically approved class of antimicrobial agents that classically function through the inhibition of bacterial cell‐wall biosynthesis by sequestration of the precursor lipid II. The oxidative crosslinking of the core peptide by cytochrome P450 (Oxy) enzymes during GPA biosynthesis is both essential to their function and the source of their synthetic challenge. Thus, understanding the activity and selectivity of these Oxy enzymes is of key importance for the future engineering of this important compound class. Recent reports of GPAs that display an alternative mode of action and a wider range of core peptide structures compared to classic lipid II‐binding GPAs raises the question of the tolerance of Oxy enzymes for larger changes in their peptide substrates. In this work, we explore the ability of Oxy enzymes from the biosynthesis pathways of lipid II‐binding GPAs to accept altered peptide substrates based on a vancomycin template. Our results show that Oxy enzymes are more tolerant of changes at the N terminus of their substrates, whilst C‐terminal extension of the peptide substrates is deleterious to the activity of all Oxy enzymes. Thus, future studies should prioritise the study of Oxy enzymes from atypical GPA biosynthesis pathways bearing C‐terminal peptide extension to increase the substrate scope of these important cyclisation enzymes.


Introduction
The glycopeptide antibiotics (GPAs) are a fascinating class of nonribosomal peptide natural products, possessing complex structures and an equally impressive biosynthesis. [1,2] Central to this is the role of a nonribosomal peptide synthetase (NRPS) assembly line that generates the linear peptide core, [3] and the subsequent activity of a cascade of cytochrome P450 (Oxy) enzymes [4][5][6] that perform the signature sidechain crosslinking seen in GPAs ( Figure 1). [7,8] Whilst most reported GPAs, including both the clinical examples of GPAs (vancomycin, teicoplanin) as well as second generation semi-synthetic GPAs (oritavancin, dalbavancin) consist of heptapeptide backbones bearing three or four side-chain crosslinks that target peptidoglycan biosynthesis by inhibiting the incorporation of lipid II, a major effort from the Wright group has shown the diversity of GPAs extends beyond lipid II-binding GPAs into the so-called type V GPAs. [9][10][11][12] Type V GPAs, exemplified by complestatin, [13] kistamicin [4] and more recently corbomycin, [11] show significant differences in structure to lipid II-binding GPAs, including a lack of glycosylation, altered peptide backbones (including extension beyond heptapeptides) and different crosslinking patterns within their aglycones ( Figure 1). The revelation that type V GPAs act through a new mechanism of action to target bacterial cell wall biosynthesis (via autolysin inhibition) [11] and that the origin of the amino acid selection (adenylation, A-) domains within their NRPS assembly pathways differs from lipid II-binding GPAs shows that these peptide natural products should arguably be considered a different family of antimicrobial peptides. [12] Despite these differences, the key biosynthetic and structural feature of GPA biosynthesis-the complex, Oxy-mediated crosslinking of the linear peptide-is maintained across all GPAs studied to date. [8,[15][16][17] Indeed, a previous investigation of the crosslinking cascade found in the biosynthesis of the type V GPA kistamicin showed that this process remained linked to peptide biosynthesis on the NRPS assembly line through the recruitment of the Oxy enzymes via an X-domain. [14] This domain-unique to GPA biosynthesis-interacts with the Oxy enzymes and delivers the adjacent peptidyl carrier protein (PCP)-bound peptide substrate to these cyclisation enzymes and appears conserved across all GPAs. [17] Differentiation of the mode of action of the Oxy enzymes also appears to have emerged in type V GPA biosynthesis, with kistamicin differing from the paradigm established for the lipid II-binding GPAs in that a single Oxy enzyme was now demonstrated to install more than one crosslink in the final peptide structure. [14] Having seen that the general mechanism of Oxy activity and recruitment via an interaction platform is conserved in type V GPA biosynthesis, the question next arose as to how the structural changes in the peptides seen with type V GPAs might be tolerated by Oxy enzymes from lipid II-binding GPA biosyn-thesis pathways. Previous studies have shown that alterations to the peptide substrates of the GPAs can be tolerated at specific positions within the peptide, [18,19] and further than divergent crosslinking patterns and Oxy enzyme behaviour can be generated using significantly altered linear precursor peptides. [20] Alterations to the length of the linear precursor peptide in pioneering in vivo work from the Süssmuth group, [21] together with in vitro studies using β-amino acids within the precursor peptide [22] have also shown that there were likely to be challenges with alterations in the peptide structure at the C terminus, but the effect of alterations at the N terminus of the peptide were unknown. Given this, we undertook to explore the effect of altered peptide substrates on the cyclisation ability and examples of the structures from different GPA classes together with their respective crosslinking machineries. A) Nonribosomal peptide synthetase (NRPS)-mediated synthesis of the linear vancomycin peptide, followed by side-chain crosslinking catalysed by cytochrome P450 (Oxy) enzymes. The peptide is further modified after cleavage from the NRPS. Vancomycin affects its antibiotic activity by binding to the C terminus of the peptidoglycan precursor lipid II (d-Ala-d-Ala), which is enabled by its crosslinked structure. B) Structures of different GPAs with crosslinks installed through the side chains of aromatic amino acids by different Oxy enzymes. C) The stereochemistry of the amino acids in GPA sequences represented as a table. Key for NRPS domains: A: adenylation, C: condensation, E: epimerisation, X: cytochrome P450 recruitment, TE: thioesterase, PCP: peptidyl carrier protein, the phosphopantetheine moiety is shown as undulated line. Oxy enzymes: Oxy B: red, Oxy A: green, Oxy C: orange, Oxy E: blue; Hpg: 4-hydroxyphenylglycine,*: stereochemistry unknown. of Oxy enzymes from the biosynthesis of lipid II-binding GPAs as a means both to explore the tolerance of lipid II-binding GPAs for alterations to their core peptide structures and to identify potentially interesting Oxy enzymes from type V biosynthesis for future characterisation.

Results and Discussion
Extended peptide design and synthesis The central heptapeptide comprising d-Leu1 to l-3,5-dihydroxyphenylglycine (Dpg)7 remained common to all of the vancomycin-like peptides tested in this study, with extensions of the sequence made at either the N or C terminus. Peptide design was motivated both by the nature of the amino acid and its stereochemistry to probe the cyclisation cascade using Oxy enzymes from lipid II-binding GPA biosynthetic pathways. Based on previous results, l-and d-Arg were chosen for inclusion at position 0 and 8 as this amino acid has been shown to be poorly tolerated in position 3 of the peptide, whilst being well accepted in position 1. [18] The C-terminal modification of vancomycin with l-Arg is particularly attractive since such a simple modification in previously synthesised derivatives has showed antimicrobial activity towards Gram-negative bacteria. [23] The modification of vancomycin at the C terminus has been shown in several studies to have a minimal impact on antimicrobial activity and consequently, we also explored the incorporation of propargylglycine (l-and d-Pra) since this amino acid is relatively small, nonpolar/nonreactive under P450 cyclisation conditions, and further offers a handle for modification postcyclisation. The acceptance of such amino acids in GPA biosynthesis could be beneficial in selective modification with further payloads. [24] However, modifying the C terminus of the linear GPA precursor peptide has been associated with a loss P450 activity, particularly with the second enzyme (OxyA) in the P450 cyclisation cascade that forms the D-O-E ring, [22] which has also been shown to be less stable than OxyB in certain cases. [25] In contrast to modification of the C terminus, modifications at the peptide N terminus appear to be more widely tolerated since it has been shown that the incorporation of aromatic residues such as those found in teicoplanin [26] or positively charged residues can be tolerated. [18] In addition to l-and d-Arg, we also explored the incorporation of l-and d-4hydroxyphenylglycine (Hpg) at position "0" of the peptide to provide substrates for the Oxy cyclisation cascade that are structurally related to newly identified examples of type V GPAs. [10] Based on these considerations, we therefore chose to synthesise a vancomycin-like heptapeptide (1) as a control and eight further extended octapeptides related to vancomycin (  Schematic workflow commencing with peptidyl-CoA synthesis in two steps: i) solid-phase synthesis of peptide hydrazides on 2-chlorotrityl chloride resin (Trt); ii) conversion of peptide hydrazides in peptidyl-CoAs. Subsequent enzymatic loading of these peptides onto a PCP-X-didomain construct by using the promiscuous phosphopantetheinyl transferase Sfp was followed by in-vitro enzymatic peptide cyclisation using the Oxy enzymes prior to cleavage of the loaded peptides and HRMS/MS 2 analysis. The sequences of peptides 1-9 are shown in the table; R 1 = H unless otherwise indicated; R 2 = S-CoA (also present at the C terminus of extended peptides 2-5). For key, see Figure 1. exploited solid-phase synthesis based on Fmoc-chemistry using optimised conditions for amino acid coupling and Fmocdeprotection, together with the adoption of a hydrazide oxidation route to generate thioester conjugate peptidyl-CoAs. [27] Once the peptidyl-CoAs were obtained, chemoenzymatic P450-mediated cyclisation was performed in three steps ( Figure 2): 1) peptidyl-CoA loading onto the PCP-X di-domain protein; 2) sequential P450-catalysed crosslinking reactions and 3) cleavage of the crosslinked peptides from the PCP-X construct and analysis by LCMS. [18] The products were further characterised by LC-HRMS and MS 2 experiments to explore the location of the rings installed by the Oxy enzymes and to characterise the common fragments of these cyclised peptides.

Octapeptide turnovers and analysis
From previous studies, it has been shown that the formation of three crosslinks between aryl side chains at residues 2-4, 4-6, and 5-7 within vancomycin-type GPAs occurs through the activity of Oxy enzymes in the following order: 1) initial C-O-D ring formation (performed by OxyB), 2) subsequent D-O-E ring formation (performed by OxyA), and 3) finally biaryl A-B ring formation (performed by OxyC). [4][5][6]17,[28][29][30][31][32][33][34] OxyE is present in type III-IV GPAs and installs the F-O-G ring between residues 1 and 3, with this occurring after OxyB activity but before that of OxyA. [26,35] A comparable OxyE enzyme has been identified in corbomycin biosynthesis, although the order of activity in this case is as yet unclear. [11] This pathway is further complicated by the possibility of type V OxyC enzymes to install both links normally installed by OxyB and OxyC in type I-IV GPAs, [14] as well as evidence from in-vivo studies of complex mixtures of crosslinked peptides resulting from Oxy deletion. [29] Commencing with turnovers of the control peptide 1, we observed a net loss of 2 (  fragmentation of the bicyclic peptide 1 c indicates the formation of both C-O-D and D-O-E rings, whilst the loss of all the Hpg, ClTyr and Dpg ions was noted in the formation of the tricyclic peptide product 1 d. Such MS 2 fragmentation analysis allows the assignment of the products of Oxy turnover, and a complete MS 2 analysis is shown in Figures S2-S5, highlighting common fragments for monocyclic and bicyclic products. Turning next to the turnover of peptides 2-5 (with extended C termini), our analysis clearly demonstrates that extension of the C terminus is not compatible with effective enzymatic crosslinking by Oxy enzymes from lipid II-binding GPA biosynthesis clusters (Figures 4 and S10-S29). Indeed, the activities of all Oxy enzymes were significantly reduced by the presence of the extended residue at position 8. The conversion of linear peptide to the monocyclic product by OxyB was halved for 2 compared to the control, with OxyB barely functional for 3-5. The low conversion seen for OxyB was further mirrored by greatly reduced functionality of OxyA for the second cyclisation step. OxyC activity was extremely low for all four peptides, which is due to this enzyme requiring the effective activity of both OxyB and OxyA to provide substrate for this enzyme. The stereochemistry present in position 8 of the peptide seems to be relevant for OxyB activity, with d-configured residues added in this position resulting in less than half the conversion compared with the l-amino acid. Curiously, the OxyC con-version of 2 did not follow this trend, thus suggesting that alterations in the stereochemistry of the terminal peptide residue can affect different Oxy enzymes in a substrate dependent manner. These results indicate that extension at the peptide C terminus is generally challenging for Oxy enzymes from lipid II-binding GPAs, and further suggests that this extension is interfering with the correct placement of the linear peptide present on the PCPÀ X didomain in the active sites of the Oxy enzymes.
Turning to peptides 6-9, the Oxy enzymes demonstrated a greater tolerance for N-terminal extended peptides, as the three cyclic products were formed at the same conversion yield as control 1 (Figures 4 and S30-S53). Both OxyB and OxyA enzymes appear far less selective when it comes to the stereochemistry of the extra residue at the N terminus, which again suggests the importance of maintaining the C terminus of the peptide as the site of attachment to the PCP-X didomain to ensure correct peptide placement for the Oxy enzymes. Based on the MS 2 analysis, the additional N-terminal Hpg residues were not found to participate in crosslink formation. OxyC activity towards peptides 6-9 was more variable than for OxyB/ OxyA and displayed more of an apparent stereochemical preference for L-configured residues at position 0 of the peptide. Indeed, the reduced activity for OxyC when compared to the improved OxyA activity for these peptides was most curious, and we sought to understand this by closer investigation of the bicyclic products produced in these reactions to determine whether this situation could be explained by competition between these enzymes.

Detection of an unexpected bicyclic product
Oxy activity towards non-native peptide sequences have been widely reported, and particularly for OxyB [20,36,37] and the dual functional OxyC enzyme from kistamicin biosynthesis. [14] Given this, a closer investigation of the MS 2 fragmentation patterns of the products from the turnovers of peptides 6-9 revealed that alternate cyclisation patterns appeared to be present in the turnover of peptide 8. We observed multiple product peaks and a different MS 2 fragmentation pattern compared to the common C-O-D + D-O-E bicyclic fragments at a retention time of 45.77 min. To identify the structure of the bicyclic product 8 e from the turnover of 8, we conducted HRMS 2 for this product ([M + H] + 1261.3808), which revealed an unexpected bicyclic pattern was present in 8 e. Analysis strongly supports a bicyclic peptide containing both C-O-D and A-B rings, which is revealed by the presence of major peaks corresponding to calculated b3 and y2 ions (together with the loss of NH 3 and further loss of CO; Figure 5). Close inspection of the turnover reactions performed with other all peptides showed that the b3 and y2 ions identified for 8 were not observed in the other reactions, suggesting that this bicyclisation reaction is related to the structure of 8. Whilst MS 2 fragments cannot distinguish between A-B and A-O-B ring systems, previous experiments have indicated that OxyC enzymes from lipid II-binding GPA biosynthesis pathways exhibit a preference for A-B ring formation despite changes to the structure of the peptide. This suggests that the ring found in 8 e is likely to be an A-B ring. [33,38] To ascertain the origins of this unusual bicyclic peptide species, we next attempted turnovers of 1 and 8 with different combinations of Oxy enzymes present (OxyB alone, OxyB + OxyA and OxyB + OxyC). Our initial analysis of the MS 2 data from these different Oxy enzyme combinations looked for the loss of Hpg, Dpg or Cl-Tyr fragments to determine the site of cyclisation in the products observed. As expected, LC-HRMS analysis of the turnover of both 1 and 8 by OxyB alone showed high levels of C-O-D monocyclic product formation ( Figures S6  and S46), and the anticipated C-O-D/D-O-E bicyclic peptide when OxyB and OxyA were included (Figures S7 and S47). However, when OxyB and OxyC, were included in turnover assays, we found MS 2 fragments indicating that cyclisation was affording the unusual bicyclic products 1 e and 8 e (Figures S8  and S48). From these experiments, formation of the tricyclic product (with the final A-B ring installed by OxyC) only occurs from the bicyclic species containing the C-O-D and D-O-E rings. Once the unusual bicyclic compound (containing the C-O-D and A-B rings) has been formed, OxyA appears unable to form the D-O-E ring. This order of ring formation appears conserved and agrees with that seen for the type V GPA kistamicin, where a sole OxyC enzyme installs both C-O-D and A-O-B rings but requires OxyA activity between these two steps. 14 Having seen that OxyC activity can occur after OxyB activity, we explored whether such effects were also seen in Oxy deletion strains of the producer of the vancomycin-type GPA balhimycin. Whilst such a product was not reported in previous studies, [4][5][6]30] comparable experiments performed with the type IV GPA A47934 had shown that OxyC could function when OxyA was deleted from the producer strains. [29] Following cultivation of the three balhimycin deletion strains Amycolatopsis balhimycina ΔoxyB, ΔoxyA and ΔoxyC, analysis of culture filtrates of these deletion strains showed the anticipated linear (10 a) and C-O-D/D-O-E bicyclic (10 c) products from the ΔoxyB and ΔoxyC strains respectively (Figures S54-S56). Close analysis of the products of the ΔoxyA strain showed that whilst the monocyclic C-O-D peptide 10 b was the dominant product, there was also the formation of a bicyclic peptide product in this strain, suggesting that OxyC can act on monocyclic peptides in such type I GPA cyclisation pathways if OxyA is missing. Taken together, these experiments suggest that the formation of 8 e is due to the activity of OxyC in place of OxyA after OxyB has formed the C-O-D crosslinked peptide 8 b, with this the first example of such a competitive bicyclisation when a functional OxyA enzyme is also present and able to cyclise the monocyclic precursor peptide.

Conclusions
Our investigation of the tolerance of the Oxy cyclisation enzymes taken from lipid II-binding GPAs for alterations to their peptide substrates suggests that whilst peptide extension is tolerated relatively well at the N terminus, extensions at the peptide C terminus are highly deleterious to effective cyclisation. Our results show that alterations in peptide structure can also lead to situations where OxyA and OxyC begin to compete for the same substrate, which raises the question of whether the bifunctional activity of some OxyC enzymes in type V pathways is controlled by thioesterase domain selectivity for different cyclic peptide products as opposed to innate Oxy selectivity in these systems. Given the recent reports of type V peptide structures that diverge from the "typical" heptapeptide core of lipid II-binding GPAs, our results suggest that exploring the Oxy enzymes from systems bearing a C-terminal peptide extension -such as corbomycin -will be valuable if we are to understand how effective cyclisation of extended peptides can be maintained in these systems. Such data will be essential if we are to exploit the Oxy enzymes as more general biocatalysts for peptide cyclisation as well as engineering the production of new type V GPA derivatives.

Synthesis of peptidyl-CoAs:
Peptidyl-CoAs were synthesized manually on solid phase at 0.05 mmol scale with subsequent hydrazide activation and displacement to generate the desired peptidyl-CoA thioesters. Fmoc-protected amino acids were synthesised and purified according to the protocols previous reported. [39] Briefly, 2-chlorotrityl chloride resin (200 mg) was swelled in DCM (8 mL, 30 min), washed with DMF (3 ×) and incubated with a 5 % hydrazine solution in DMF (6 mL, 2 × 30 min). The resin was washed with DMF (3 ×), and a solution of DMF/TEA/MeOH (7 : 2 : 1; 4 mL, 15 min) added to cap unreacted 2-chlorotrityl groups. The first Fmoc-protected amino acid (1 equiv., 0.05 mmol) was coupled to the resin overnight using COMU (1 equiv., 0.05 mmol) and 2,6lutidine (0.12 M in DMF, 3 mL). Unreacted hydrazine groups were then capped with Boc-glycine (3 equiv., 0.15 mmol) that had been activated prior to addition using COMU (3 equiv., 0.15 mmol) and 2,6-lutidine (0.12 M in DMF, 3 mL) for 1 h. Subsequent Fmoc removal was performed using 1 % DBU in DMF (3 mL, 3 × 30 s) followed by coupling of the desired Fmoc-amino acid (0.15 mmol) after pre-activation with COMU (3 equiv., 0.15 mmol) and 2,6lutidine (0.12 M in DMF, 3 mL) for 1 h. Cleavage of the hydrazide peptide from the resin and removal of side chain protecting groups was accomplished using TFA/TIPS/H 2 O (95 : 2.5 : 2.5 v/v/v, 3 mL) with shaking at room temperature for 1 h (extended to 2 h for argininecontaining peptides). The resin was removed by filtration and washed with TFA (2 ×) and DCM. The filtrate was concentrated under a stream of N 2 to~1 mL, the peptide precipitated with icecold diethyl ether (~9 mL) and collected by centrifugation in a flame-resistant centrifuge (Spintron GT-175). The crude peptides were purified using preparative RP-HPLC (10-50 % MeCN gradient over 40 min). Purified hydrazide peptides were dissolved in buffer A (6 M urea and 0.2 M NaH 2 PO 4 , pH 3) to a final concentration of 5 mM. The solution was cooled to À 15°C using a salt/ice bath, before addition of 0.5 M NaNO 2 (0.95 equiv.), with the mixture stirred for 10 min. CoA (1.2 equiv., dissolved in buffer A) was added to the reaction and the pH slowly adjusted to 6.5 using KH 2 PO 4 / K 2 HPO 4 buffer (6 : 94, v/v, 1 M, pH 8.0). The mixture was stirred at À 15°C for 2 h before the peptidyl-CoA was purified using preparative RP-HPLC (10-50 % MeCN gradient over 40 min). All purifications were performed using a Shimadzu high performance liquid chromatography system equipped with an SPD-M20A Prominence photo diode array detector and two LC-20AP pumps. Preparative separations were performed using a Zorbax SB-C 18 column (Agilent, 7 μm, 21.2 × 250 mm) using a flow rate of 10 mL min À 1 ; mobile phases used were water + 0.1 % TFA (solvent A) and HPLC-grade ACN + 0.1 % TFA (solvent B). Peptide analysis was conducted on an HPLC-MS system from Shimadzu (LCMS-2020, ESI operating in positive and negative mode); mobile phases used were water + 0.1 % FA and ACN + 0.1 % FA. For LCMS analysis, see Figures S57-S64.
Enzymatic cyclisation reactions: Cytochrome P450-mediated peptide crosslinking was performed in three steps: 1) peptide loading onto the PCP-X tei protein; 2) P450-catalysed crosslinking; and 3) cleavage of the crosslinked peptides from the PCP-X tei construct.
Crosslinked peptides were subsequently cleaved from the PCP-X tei didomain by the addition of a 40 % methylamine solution in water (0.5 M) with incubation for 15 min at room temperature. After cleavage, 850 μL of 0.1 % FA in water was added into the reaction to adjust the pH to~7. Purification was performed using solid phase extraction (SPE) column (Bond Elut Plexa 30 mg/mL, Agilent Technologies) that had been activated with 0.1 % FA in MeOH (1 mL) and equilibrated with 0.1 % FA in water (1 mL). The neutralised solution (1 mL) was applied to the equilibrated SPE column via gravity flow, washed with 0.1 % FA in water (1 mL) and eluted with 1 mL of 0.1 % FA in ACN/H 2 O (50 : 50). The sample was flash frozen with liquid nitrogen and dried using a freeze-dryer (Christ Alpha 1-2 LD plus) and dissolved in 50 μL of 0.1 % FA in ACN/H 2 O (5 : 45) prior to analysis by HRMS.
Cultivation of Δoxy strains and peptide isolation: One square centimetre of Amycolatopsis balhimycina ΔoxyB, ΔoxyA and ΔoxyC [30] R5-agar cultures were scraped off and used to inoculate a preculture of 50 mL R5 medium. [43] Three-day precultures (5 mL) were then used to inoculate the main cultures of 100 mL R5 medium. The liquid cultures of A. balhimycina ΔoxyB, ΔoxyA, ΔoxyC were cultivated in an orbital shaker (180 rpm) in 500 mL Erlenmeyer flasks with one baffle and steel springs at 29°C for 5 days. Culture broths were separated from the mycelia by centrifugation and used for HPLC-MS analysis.
HRMS analysis: Samples were separated on a RSLC 3000 LC system (Thermo) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). The LC system consisted of a trap column Acclaim PepMap 100 (100 μm × 2 cm, nanoViper, C 18 , 5 μm, 100 Å; Thermo Scientific) and an Acclaim PepMap RSLC analytical column (75 μm × 50 cm, nanoViper, C 18 , 2 μm, 100 Å; Thermo Scientific). Samples were loaded onto the trap column in μL-pickup mode using 2 % acetonitrile, 0.1 % TFA loading buffer. Using a 30 min gradient and a flow rate of 250 nL min À 1 , compounds were eluted from the column by increasing concentrations of buffer B (80 % acetonitrile, 0.1 % formic acid (FA); ranging from 6 % to 30 %), and ionised in the nanospray source operated at 1.7 kV. The mass spectrometer was operated in both data-dependent acquisition (DDA) and PRM mode to target the appropriate species. Full ms1 scans were acquired at 240.000 resolution. MS 2 spectra were acquired at 15.000 resolution (for both DDA and PRM) with a 1.4 m/ z isolation window. Higher-energy Collision Dissociation (HCD) was used for fragmentation using a fixed normalized collision energy (NCE) of 24 in case of DDA, and a stepped NCE of 22, 26 and 30 in case of PRM. Raw data was manually analysed in XCalibur QualBrowser (Thermo Scientific), with extracted ion chromatograms to the predicted species generated with 10 ppm mass tolerance. MS 2 spectra corresponding to the predicted mass were manually characterised for ring closures based on predicted neutral loss peaks of non-crosslinked residues.
Calculation of turnover yields: Results are presented as the percentage conversion of a specific peptide relative to the amount of peptide substrate present, bearing in mind that substrates can been formed in the previous cyclisation step by another Oxy enzyme and that the product of a specific Oxy can also be converted further by additional Oxy enzymes. The Oxy activities were calculated as follows: All experiments were performed as triplicates (except for 1, 4 and 5 which were performed in duplicate) followed by calculation of average and standard deviation.