C T Chen

W J Su

W J Wu

D Vasic

F Costa

Meso-scale piezoelectric energy harvester for low-frequency rotational motion

Keywords: frequency up-conversion, piezoelectric energy harvesting, wind turbine, magnetic plucking

Wind energy has been seen as a most potential renewable energy. However, in shore environments, the wind turbine usually suffers constantly impact of sand and strong wind speed, which causes the surface deteriorate: cracks may appear. To reduce the wind turbine operations and maintenance (O&M) cost, assembling a self-powered surface condition monitoring system (SCMS) becomes the most important measures. In this paper, a meso-scale piezoelectric energy harvester (PEH) was fabricated, based on a tapered cantilever beam to scavenge the rotational energy to power SCMS. The advantages are to increase its output power density and its lifetime comparing to the traditional rectangular cantilever beam. A frequency up-conversion method was adopted to accommodate PEH to working under variety of rotational speed by using two opposing magnets. With different distances between two magnets, the output voltage and the daily output energy of the PEH were investigated under 5 rpm -30 rpm rotational speed. The maximum output voltage is 2.7 V, 9.1 V and 13.6 V when the magnets spacing is 3 mm, 2 mm and 1 mm, respectively. For the magnet spacing of 1 mm, the daily output energy of the PEH was estimated to be 5.26 J under periodic magnetic plucking at 30 rpm, much higher than the 0.2 J of SCMS's daily energy consumption, making this harvester an excellent solution for the abovementioned needs.

INTRODUCTION

In recent years, the demand for electric energy has increased year by year. To the countries who heavily rely on the imported fossil fuels will cost huge economic and environmental burden. Among them, Taiwan is one of the countries in that situation. Fortunately, the consultancy company 4C Offshore reported that Taiwan possess 80% of the world best wind field which is located on Taiwan strait [START_REF] Offshore | Global Wind Speed Rankings[END_REF]. The government makes a big push to extract international investment on the development of wind energy technology especially after the Fukushima disaster highlighted the risks of using nuclear power in the region prone to earthquake. However, because of the geographical location and narrow coastal terrain, wind turbines frequently suffer strong wind containing high sand concentration in summer. Both of them are the risks to destroy wind turbine blade. As time goes on, the destruction of the wind turbine may occur starting from its surface of the blade. To reduce the operations and maintains (O&M) cost, assembling a self-powered surface condition monitoring system (SCMS) becomes the most important task. With the help of SCMS, we can monitor the surface condition of the wind turbine blade and schedule the maintenance in advance. For the proposed self-powered SCMS shown in Figure 1, a mesoscale piezoelectric energy harvester (PEH) with its interfacing circuit are responsible to generate sufficient electric energy to supply the ultrasonic transducer of SCMS. Finally, the data from SCMS will be transferred back to information center through a wireless module.

In this paper, we will focus on the design, fabrication and the validation of the PEH in energy harvesting system.

Since wind turbines have varying and low rotational speed (5 rpm to 20 rpm) depending on the wind condition, the widely used resonant type of PEH is not available for this application. Band broadening and frequency up-conversion method are the common solutions to accommodate PEH working at its off-resonant frequency. There are some researchers using direct impact [START_REF] Fang | A music-box-like extended rotational plucking energy harvester with multiple piezoelectric cantilevers[END_REF] or nonlinear magnetic plucking[] to improve the performance of PEH at low rotational speed. H. Fu. et al. presented theoretical and experimental validation of bi-stability and frequency up-conversion mechanism of a rotational PEH by using two external repelling magnets [START_REF] Fu | Rotational energy harvesting using bi-stability and frequency up-conversion for low-power sensing applications: Theoretical modelling and experimental validation[END_REF]. The unimorph PEH is 33.5mm in length, 2mm in width and 0.25 mm in thickness direction. The results indicate the maximum power in double-wall mode is around 54W at around 11 Hz rotational frequency. P. Pillatsh et al. designed a 30 mm package containing a rotational PEH with dimension of 19.5 mm  1 mm  0.37 mm for human body application [START_REF] Pillatsch | A piezoelectric frequency up-converting energy harvester with rotating proof mass for human body applications[END_REF]. The PEH provides maximum output power with a peak value of 43 W at 2 Hz and 20 m/s 2 rotational speed and acceleration level, respectively. Even though there are lots of research on it, the rotational speed is much higher than the wind turbine application. The rotational speed of wind turbine is around 5 rpm to 20 rpm corresponding to 0.08 Hz to 0.3 Hz. Besides, the average power is positively related to the rotational speed. Thus, the design of frequency up-conversion mechanism and the PEH device are the main challenges in this paper.

Figure 1 The proposed self-powered surface condition monitoring system This paper is divided into three parts. The first part is the equivalent nonlinear model for rotational PEH. First part is the nonlinear model for energy harvesting system including the PEH and the nonlinear magnetic force. Then, it goes to the fabrication process of PEH and the brief power consumption of the self-powered SCMS system. The experimental setup and experimental results of PEH in different shape, with different magnet spacing and at different rotational speed will be shown in the last part.

MODEL AND FABRICATION PROCESS 2.1 Model

Figure 2. Schematics diagram of rotational energy harvesting system

The nonlinear model of PEH excited by magnetic plucking can be divided into two parts. One is the nonlinear mechanism of piezoelectric energy harvester and the other is the nonlinear magnetic force. Consider a unimorph PEH with a magnet A at its free end clamped on a revolving disc with varying rotational speed 𝑉 𝑟 . There is another driving magnet B is fixed on a stationary disc as shown in Figure 2. The driving magnet B is periodically applying a repelling force on magnet A with the closest distance dmag between magnet A and B. The governing equations of the nonlinear PEH can be described as following equations [START_REF] Fang | Nonlinear dynamic analysis of cantilevered piezoelectric energy harvesters under simultaneous parametric and external excitations[END_REF] and its equivalent model is shown as Figure :

𝑚𝑥̈+ 𝐹 𝐷 + 𝐹 𝑠 = 𝐹 𝑚𝑎𝑔 -𝐹 𝑃 (1)
-𝜃𝑥̇+ 𝐶 𝑝 𝑉 ̇+ 𝑉 𝑅 𝐿 = 0

(2)

𝐹 𝐷 = 𝑐 1 𝑥̇+ 𝑐 2 𝑥 2 𝑥̇ , 𝐹 𝑠 = 𝑘 1 𝑥 + 𝑘 2 𝑥 3 , 𝐹 𝑃 = 𝜃𝑉 (3)
Where 𝐹 𝑚𝑎𝑔 is the magnetic force from driving magnet B, m is the mass of magnet A, x is the tip displacement of the beam, 𝑥̇ is the velocity, 𝑥̈ is the acceleration, c1 and c2 are linear and nonlinear damping ratio, k1 and k2 are the terms of linear and nonlinear stiffness, 𝜃 is the piezoelectric coupling factor, V is the voltage across the load resistance RL, Cp is the clamped capacitance of piezoelectric.

Figure 3 Equivalent model of nonlinear PEH excited by magnetic plucking

In order to calculate the magnetic force when the driving magnet B crossing to magnet A, we evaluate the magnetic force distribution while the closest distance dmag is 1 mm, 2 mm and 3 mm. The magnetization of two magnets is 0.73 T. The hollow data points in Figure show the normalized magnetic force distribution from simulation. We can describe it by a two Gaussian functions of x as shown in equation ( 4).

𝐹 𝑚𝑎𝑔 = 𝐴 𝐵√2𝜋 {𝑒𝑥𝑝 (- (𝑥 -𝑤) 2 2𝐵 2 ) -𝑒𝑥𝑝 (- (𝑥 + 𝑤) 2 2𝐵 2 )} (4)
where A, B and w are fitting parameters, x is the magnet spacing in out-of-plane direction as shown in Figure .   The solid lines in Figure are the curve fitting results and the values of fitting parameters A, B and w are shown in Table 1.

The results indicate the peak value of magnetic force is decreasing when the dmag is increasing. The value of the fitting parameter w is corresponding to the absolute x-axis value of the two peaks. With the shortening of the dmag, the w value is slightly increasing. Besides, because the magnetic force in this application is periodic, x can be represented as eq. ( 5).

𝑥 = 𝑥 0 + 2𝜋𝑟 • 𝑉 𝑟 60 • 𝛿(𝑡 -𝑇 𝑟 ) (5)
𝑇 𝑟 = 60 𝑉 𝑟 ⁄ (6)
Where 𝑥 0 is the initial position relative to the driving magnet B, 𝑉 𝑟 is the rotational speed and 𝑇 𝑟 is the rotational period.

Figure 4 The normalized magnetic force with different magnet spacing

Fabrication process

From our previous results of the PEHs under base excitation, the tapered shape of PEHs show outstanding output performance and better durability comparing to the widely used rectangular shape of PEHs [START_REF] Chen | The output power improvement and durability with different shape of MEMS piezoelectric energy harvester[END_REF]. In this paper, the magnetic force acts as a body load on the tip magnet which is similar to the behavior of external force under base excitation. Therefore, the tapered shape of PEHs are expected to have better output performance and durability. In this study, Lead zirconate titanate (PZT) thick film was used as the active material due to its great piezoelectric properties.

The geometrical parameters of each device are shown in Table 2. The width of the free end of PEH is designed as 0 mm (tapered shape) and 6 mm (rectangular shape), respectively. All the dimensions of devices except the width of the free end are kept constant. A neodymium magnet with dimension 3.8 mm × 3.8 mm × 2 mm (length  width  thickness) is bonded on the tip of each PEH. First of all, the pre-cleaning process of 301 stainless steel should be done to remove the metallic oxide and organic residues on the surface. After the pre-cleaning process, the 9 µm thick PZT layer is deposited on the steel by aerosol deposition method. Around 220 nm Pt/Ti layers is then deposited on the top of PZT film as the top electrode of PEH devices. The beam shape was defined with photolithography and released with wet etching by using aqua regia solution. Finally, all the devices are annealed and the magnet is attached on the tip of each beams.

DAILY ENERGY CONSUMPTION OF SCMS

Figure 7 The self-power surface condition monitoring system

The self-powered SCMS building blocks are shown as Figure . Typically, the self-powered system is very low-duty working and therefore the energy harvesting interfacing circuit has to turn on all the time and to harvest as much power as possible into capacitor. To have a brief estimation of daily energy consumption of this system, we suppose that there is only one measurement in a day and each measurement takes one second. For the SCMS, a commercial voltage amplifier LT3469 is selected to amplify the output voltage from the capacitor to drive the ultrasonic actuator in SCMS. In order to have an obvious sensing signal into signal processing, a low noise signal amplifier AD8608 is selected. A Bluetooth low energy module of nRF51822 is used to send the data back to the information center. The daily energy consumption is around 0.2 J.

EXPERIMENTAL SETUP AND RESULTS

Experimental setup

Figure 8 shows the experimental setup of rotational PEH excited by magnetic plucking. The revolving disc is fixed on the motor controlled by a laptop and a motor controller. The range of rotational speed is set from 5 rpm to 30 rpm which generally covers the typical rotational speed of wind turbines. The PEH is clamped and fixed on the revolving disc and the driving magnet B is attached on another stationary base. The output voltage and output power are measured by connecting PEH to its optimal impedance of 100 kΩ. Since the input voltage range of the data acquisition (DAQ) is from -10V to +10V, the output voltage is connected to a voltage divider before getting into DAQ.

Figure 8 Experimental setup for rotational PEH From the magnetic force distribution profile shown in Figure , the magnetic force is increasing (i.e. increasing the output performance of PEH) with decreasing magnet spacing. However, the durability of PEH becomes a very important issue especially for the wind turbine application. While considering the durability of PEHs, the magnetic force cannot be increased endlessly to destroy the piezoelectric layer. Thus, the following discussion will focus on the output performance and durability of PEH excited by different magnitude of magnetic force. The output voltage profile can be divided into three parts that are magnet B approaching to magnet A, magnet B crossing magnet A and the beam free-oscillating region. For the first part, the output voltage slightly decreases when the magnet B approaching and repelling magnet A. For the second part, the magnet B releases the beam in a short period and quickly crossing by magnet A. Finally, the magnet B leaves from magnet A and the PEH starts oscillating at its natural frequency without any external magnetic force. By mean of magnetic plucking, PEHs can be operated at its resonant frequency by using an offresonant external force which is known as frequency up-conversion method.

Different magnet spacing and rotational speed

For the rotational speed at 5 rpm, the maximum output voltage is 13.58 V, 9.10 V and 2.73 V for magnet spacing at 1 mm, 2 mm and 3 mm, respectively. The subplot in Figure also indicates the voltage profile is highly related to the magnetic force profile. Because of the narrow and sharp magnetic profile of magnet spacing at 1 mm, the output voltage not only changes dramatically in the very beginning but also achieve the highest amplitude.

Comparing to the output voltage at rotational speed from 25 rpm to 5 rpm, the maximum output is 9.60 V, 9.41 V and 9.0 V, respectively. The output voltage of PEH at higher rotational speed is slightly decreasing in the very beginning and then rapidly increasing as the driving magnet B crossing magnet A. In addition, the damping effect caused by magnetic force is more obvious in the case of low rotational speed. The damping effect will reduce the output performance during the free oscillation after the magnet B crossing to magnet A. Thus, for the comparison of the output energy from single magnetic excitation, the better performance at higher rotational speed can be expected.

Different shape of PEH beams

Secondly, we consider the output performance difference and durability by changing the shape of PEH. In order to make a fair comparison, the brand new PEHs in tapered-shape and rectangular-shape beams were used to verify the output performance. Figure shows the output voltage of PEHs with magnet spacing at 2 mm and rotational speed at 5 rpm. The maximum output voltage of PEH is -7.0 V and -5.7 V respectively in tapered shape and rectangular shape of the beam.

Figure 10 The output voltage of tapered-shape and rectangular-shape PEH

In order to quantify the output energy during single magnetic plucking, the output power was integrated in a single period.

Besides, the durability of PEHs were tested from low speed to high speed and the magnet spacing changes from 3 mm to 1 mm as shown in Figure . Although both of PEHs show stable output energy in all parameters tests, the output energy of rectangular PEH starts decreasing as the magnet spacing of 1 mm. The cause of output energy decreasing is related to the crack of top electrode under large deformation. Additionally, the magnetic damping effect was magnified while the magnet B slowly crossing by the magnet A. The damping effect at low speed mode leads to the decreasing of energy output. For the rotational speed range from 30 rpm to 5 rpm, the output energy is decreased by around 10%.

CONCLUSIONS

In this paper, a cantilever type of PEHs with tapered beam shape are used to convert rotational energy into electrical energy to power SCMS. The PEH in meso-scale is composed of a beam with dimension of 6 mm × 9 mm × 69 m (width × length × thickness) and a tip magnet with dimension 3.8 mm × 3.8 mm × 2 mm (length  width  thickness). The high quality PZT film in 9 μm thickness is deposited as the active layer by aerosol deposition method. The tapered beam shape is confirmed to improve the output energy and the durability under the large deformation. A frequency up-conversion mechanism is realized by two repulsive magnets to accommodate PEH at varying rotational speed of wind turbine. For the rotational speed from 5 rpm to 30 rpm, the output voltage is increased from 9.0V to 9.6V with the magnet spacing of 2 mm. For the case of low-speed mode, the magnetic damping effect obviously reduces the output energy when the driving magnet B crossing tip magnet A. For the magnet spacing from 3 mm to 1 mm, the maximum output voltage is increased from 2.73 V to 13.58 V. In order to evaluate the daily output energy, the practical rotational speed of the wind turbine is put into consideration. The daily output energy is estimated to be 1036.8 mJ which is much higher than the 200 mJ of daily energy consumption of self-powered SCMS, leading our new device to be a very promising solution for autonomous monitoring of wind turbines.

  Figure shows the schematics diagram of the PEHs in this paper.

Figure 5

 5 Figure 5 Schematics diagram of cantilever device

Figure 6 :

 6 Figure 6: The fabrication process of rotational PEHThe flow chart of fabrication process is shown in Figure.First of all, the pre-cleaning process of 301 stainless steel should be done to remove the metallic oxide and organic residues on the surface. After the pre-cleaning process, the 9 µm thick PZT layer is deposited on the steel by aerosol deposition method. Around 220 nm Pt/Ti layers is then deposited on the top of PZT film as the top electrode of PEH devices. The beam shape was defined with photolithography and released with wet etching by using aqua regia solution. Finally, all the devices are annealed and the magnet is attached on the tip of each beams.

Figure 9

 9 Figure 9 The output voltage of tapered shape PEH with (a) different magnet spacing and (b) different rotational speed Figure shows the output voltage of tapered-shape PEH with different magnet spacing and rotational speed.The output voltage profile can be divided into three parts that are magnet B approaching to magnet A, magnet B crossing magnet A and the beam free-oscillating region. For the first part, the output voltage slightly decreases when the magnet B approaching and repelling magnet A. For the second part, the magnet B releases the beam in a short period and quickly crossing by magnet A. Finally, the magnet B leaves from magnet A and the PEH starts oscillating at its natural frequency without any external magnetic force. By mean of magnetic plucking, PEHs can be operated at its resonant frequency by using an offresonant external force which is known as frequency up-conversion method.

Figure 11

 11 Figure 11 Output energy per magnetic plucking of (a) tapered beam and (b) rectangular beam Due to the output voltage is substantially decreased before the next magnetic excitation even though the rotational speed is up to 30 rpm. The output energy of PEH can be calculated by equation (7). 𝐸 𝐷𝑎𝑖𝑙𝑦 = 𝐸 𝑝 • ∫ 𝑉 𝑟 (𝑡) • 𝑑𝑡 (7)

Figure 12 4 . 4

 1244 Figure 12 Daily output energy of (a) tapered beam and (b) rectangular beam4.4 Practical rotational speed of wind turbineIn general, the wind turbine is operating at varying rotational speed of 5 rpm to 20 rpm. The practical rotational speed in 24 hours is shown in Figure.The minimum and maximum rotational speed of VESTAS V80/2000 wind turbine is controlled at 9 rpm and 17 rpm. The total output energy of PEH is calculated to be 1036.8 mJ through equation (7). The output energy is much higher than the 200 mJ of SCMS's daily energy consumption.

Figure 13

 13 Figure 13 The rotational speed of wind turbine blade in Taiwan (Date: 2014.01.01)

Table 1

 1 The fitting parameters of magnetic force distribution

	Magnet spacing		Fitting parameters
	(mm)	A	B	w
	1	0.7453	1.3396e-4	0.00143
	2	0.083	0.0012	0.00174
	3	0.082	9.25565e-4	0.00224

Table 2

 2 The geometrical parameters of the devices

	Symbol	Description	Value	Units
	W1	Width of fixed end	6	mm
	W2	Width of free end	0, 6	mm

AKNOWLEDGEMENTS

The authors gratefully acknowledge the facility support from the Nano-Electro-Mechanical-Systems Research Center at National Taiwan University and the funding support gratefully acknowledge the support of Ministry of Science and Technology, Taiwan through respective grants MOST108-2911-I-002-575 (Dragon Gate project).


in NosL (white sticks). As for CteB, the side chain of the Φ amino acid coordinates both the adenine and the ribose moiety, but only EpeE makes two Hbonds with the N6 and N7 of the adenine moiety.

Extended Data Fig. 2

Interactions stabilizing the T-SPASM domain and the bridging region of EpeE. ED_Figure2.jp g a, Overall structure of EpeE highlighting the bridging region (red cartoon) between EpeE domains (radical SAM domain in light blue, T-SPASM in teal). b, The bridging region (red cartoon) makes extensive Hbonds (black dotted lines) with α3' helix and α3'-α4' loop (green cartoon) (left panel). The C-terminal helix α5' is stabilized by α3' helix through H-bond and hydrophobic interactions (right panel). c, Comparison between the twitch and SPASMdomains of representative radical SAM enzymes. Similarly to twitch radical SAM enzymes (SkfB & BtrN), EpeE coordinates a single [4Fe-4S] cluster, however, the bridging region extends toward the location of AuxII cluster found in SPASM enzymes (AnSME, CteB & SuiB). The C-terminal α5' helix of EpeE, absent in twitch-domains, is displaced at the opposite side of the AuxI cluster compared to the Cterminal α6' helix of SPASM-domain enzymes AnSME and CteB which lies against the α6 helix of the TIM barrel (missing in EpeE). Only the AuxI cluster, β1'-β2' anti-parallel sheets and α2' helix positions are conserved in all enzymes. The TIM barrel domain is colored in white. The Twitch/SPASM domains are colored by protein. The AuxI and II clusters are shown in yellow and orange sticks.

Editor summary:

Peptide epimerization is a common but enigmatic post-translational modification found in antibiotics formed from ribosomally-synthesized and posttranslationally modified peptides. Now, crystallographic snapshots, spectroscopy and biochemical investigations, have provided insight into the mechanism of peptide epimerization catalyzed by radical SAM epimerases.

Peer Review Information:

Nature Chemical Biology thanks Qi Zhang and the other, anonymous reviewers for their contribution to the peer review of this work. ED_Figure1.jp g a, EpeE topology diagram showing a truncated α5/β6 TIM barrel in the radical SAM domain and T-SPASM domain with a single AuxI cluster. The binding region is depicted in bold line. The position of the [4Fe-4S] clusters (yellow and orange) and the coordinating cysteine residues (yellow) are indicated. b, Radical SAM cluster of EpeE in interaction with SAH. Residues from the conserved: GGE, GXIXGXXE, ribose, β6 and CX 3 CXΦC motifs are colored according to their domain in EpeE (radical SAM domain). The unusual His-20 residue from the CX 3 CXΦC motif is highlighted. Hydrogen-bonds are shown with black lines, radical SAM [4Fe-4S] cluster with yellow and orange sticks and SAH with green sticks. c, The CX 3 CXΦC motif of structurally characterized members of the radical SAM superfamily of enzymes. The Φ residue is: Tyr in AnSME (green sticks) and CteB (brown sticks); Phe in SkfB (yellow sticks) and SuiB (violet sticks); Met Extended Data Fig. 3 EPR, UV-visible and HYSCORE analysis of EpeE in absence or presence of SAM.

Extended Data

ED_Figure3.jp g a, Temperature dependence of EPR spectra of dithionite-reduced reconstituted samples of wildtype EpeE in the absence (left panel) or in the presence (right panel) of a 5-fold stoichiometric excess of SAM. The microwave power was adjusted at each temperature to avoid saturation effects. Spectra have been amplitude-normalized. Number of accumulations: 4. The signal of the SAM-bound [4Fe-4S] + cluster relaxes significantly faster than the one detected in the unbound form. Indeed, the former broadens at temperatures above 6 K and is no longer visible at 30 K and above (right panel) whereas the latter is still detected without significant broadening at 30 K (left panel). Such differences in the relaxation behavior of the two forms allowed us to reveal partial conversion between these forms upon addition of SAM. Indeed, a weak contribution of the unbound form is detected in the sample incubated with a 5-fold excess of SAM when measured at 30 K (right panel). b, Power saturation experiments of dithionitereduced reconstituted samples of wild-type EpeE in the absence (upper panel) or in the presence (lower panel) of a 5-fold stoichiometric excess of SAM. Peak-to-peak amplitudes between features measured as indicated by arrows on left spectra are plotted against square root of microwave power in a log-log plot (blue filled circles). The dotted line represents the non-saturation regime for which the EPR amplitude is proportional to the square root of the microwave power. Other experimental conditions: temperature, 15 K (upper panel) or 6 K (low panel), microwave power, 0.1 mW (left spectra), number of accumulations, 4. c, UV visible analysis of EpeE wild-type (upper panel) and A3mutant (lower panel). Before (grey line) and after (black line) anaerobic FeS cluster reconstitution. d, X-band HYSCORE spectra of dithionite-reduced reconstituted samples of wild-type EpeE in the presence (upper panel) or in the absence (lower panel) of a 5-fold stoichiometric excess of SAM. Only the low frequency region is shown. Experimental conditions are given in the Methods section. The low frequency region of the HYSCORE spectrum of the anaerobically reduced and reconstituted wild type enzyme in the presence of SAM displays a complex set of signals in both the (+, +) and (-, +) quadrants which can be unambiguously assigned to a hyperfine coupling to a 14 N nucleus in the intermediate coupling regime for which the isotropic part of the hyperfine coupling constant a iso is nearly equal to twice the 14 N Larmor frequency, i.e.  I ( 14 N)  1.1 MHz (upper panel). These signals are absent in the corresponding HYSCORE spectrum of the enzyme prepared in the same conditions but without SAM (lower panel).

Extended Data Fig. 4 LC-MS analysis of EpeE incubated with peptides 4, 5, 6 & 7.

ED_Figure4.jp g

Activity of EpeE with peptide 4 (a), 5 (b), 6 (c) and 7 (d) was assayed in deuterated buffer. LC-MS analysis of peptide at T0 (upper left panel) and after 90 min incubation under anaerobic conditions (lower left panel). Comparison between the mass spectrum of the substrate (upper middle panel) and the product (lower middle panel) showed a +1 Da mass increment, consistent with 2 H-atom incorporation while mass spectrum analysis of the 5'-dA is shown in right panel. Extended Data Fig. 5 LC-MS/MS analysis of the peptide Extended Data Fig. 6 Comparison between substrate-free and peptide-bound structures of EpeE.

ED_Figure6.jp g

a, Superimposition of substrate-free and peptidebound structures of EpeE. b, Close-up view showing the major structural movements including the α3'-α5' helices (indicated by arrows in panel a) of the SPASM-related domain. The substrate-free EpeE structure is shown in grey and the peptide-bound EpeE structure in pale cyan (chain A) and deep teal (chain B). Alignment of the substrate-free andbound structures using all domains (634 residues) has a r.m.s.d. of 0.78 Å, as calculated using Coot SSM.

Extended Data Fig. ED_Figure8.jp g a, Superimposition of EpeE WT in complex with peptide 5 (pale cyan) and EpeE C223A in complex with peptide 5 (bright orange; r.m.s.d. of 0.22 Å). b, Superimposition of EpeE WT in complex with peptide 5 (pale cyan) and EpeE C223A in complex with peptide 6 (green; r.m.s.d. of 0.23 Å). c, Closeup of EpeE C223A mutant active site. The peptide 5 (left panel) was built for 9 out of 11 residues (KSKENRWIL) according to the electron density. The peptide 6 (right panel) was built for all the 11 residues (KENRWILGSGH) according to the electron density. The omit maps (blue mesh) of peptide 5 (pink sticks) and 6 (purple sticks) are contoured at 3σ. SAH (green) is depicted in stick, the radical SAM [4Fe-4S] and AuxI clusters are shown as spheres. d, Structure of EpeE C223A mutant with peptide 6 in its active site. K171 and D143 are stacking H49 from peptide 6 (left panel) while, in the substrate-free WT EpeE structure, D143 and K171 have a distinct orientation stabilized by a salt bridge (right panel). e, The presence of H49 in the structure of EpeE C223A mutant with peptide 6 provided inter-chain interactions between the two enzyme subunits.

Extended Data Fig. 9 The C223 residue in the structures of wild-type EpeE and D210A mutant.

ED_Figure9.jp g a, Interactions involving D210 in the structure of EpeE with peptide 5. D210 provides key electrostatic interactions to the substrate (residues N41 and R42) and is stabilized by a polar bond with the protein residue T5. The distance between C223 and D210 is 5.08 Å. b, Orientations of C223 in the structure of the D210A EpeE mutant. The omit map (blue mesh) of C223 in chain A (left panel) and chain B (right panel) is contoured at 3σ. C223 was modeled as a persulfurated cysteine residue. In chain A (left panel), C223 adopted two orientations.

Extended Data

Introduction

Ribosomally-synthesized and post-translationally modified peptides (RiPPs) form one of the major families of natural products [START_REF] Montalban-Lopez | New developments in RiPP discovery, enzymology and engineering[END_REF][START_REF] Benjdia | Radical SAM Enzymes and Ribosomally-Synthesized and Posttranslationally Modified Peptides: A Growing Importance in the Microbiomes[END_REF] . They have recently attracted considerable interest because of their involvement in the homeostasis of the human microbiota [START_REF] Benjdia | Radical SAM Enzymes and Ribosomally-Synthesized and Posttranslationally Modified Peptides: A Growing Importance in the Microbiomes[END_REF][START_REF] Benjdia | Sulfatases and radical SAM enzymes: emerging themes in glycosaminoglycan metabolism and the human microbiota[END_REF][START_REF] Balty | Ruminococcin C, an anti-clostridial sactipeptide produced by a prominent member of the human microbiota Ruminococcus gnavus[END_REF][START_REF] Balty | Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes[END_REF][START_REF] Balskus | The Human Microbiome[END_REF] and their potential to develop innovative antibiotics [START_REF] Donia | A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics[END_REF][START_REF] Benjdia | Radical SAM Enzymes in the Biosynthesis of Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs)[END_REF][START_REF] Benjdia | Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis[END_REF] . The biosynthesis of RiPPs usually follows a simple logic with the translation of a precursor peptide containing a leader or a follower sequence [START_REF] Benjdia | Radical SAM Enzymes and Ribosomally-Synthesized and Posttranslationally Modified Peptides: A Growing Importance in the Microbiomes[END_REF] which is recognized by tailoring enzymes via a specific domain called RRE (RiPP precursor peptide recognition element) [START_REF] Burkhart | A prevalent peptide-binding domain guides ribosomal natural product biosynthesis[END_REF] . After modification, the leader sequence is cleaved off and the modified peptide secreted. If the general principles are well established, how post-translational modifications are installed and precursor peptides recognized by biosynthetic machineries remains largely elusive 1,2 .

Among the broad diversity of RiPP post-translational modifications, epimerization has been only marginally investigated. In eukaryotes, the L-to-D conversion of peptide residues is critical for the activity of various toxins, venoms and neuro-peptides [START_REF] Heck | Posttranslational amino acid epimerization: enzyme-catalyzed isomerization of amino acid residues in peptide chains[END_REF] . However, the catalytic processes underpinning these transformations are largely unknown. In prokaryotes, the combined action of a dehydratase and a reductase has been shown to result in the conversion of L-Ser and L-Thr into D-Ala and D-α-aminobutyrate, respectively [START_REF] Huo | Discovery and Characterization of Bicereucin, an Unusual d-Amino Acid-Containing Mixed Two-Component Lantibiotic[END_REF][START_REF] Lohans | Structure and biosynthesis of carnolysin, a homologue of enterococcal cytolysin with D-amino acids[END_REF] . However, not only this transformation is restricted to Ser and Thr residues but it leads to the modification of the nature and reactivity of the amino acid side-chain.

Recently, novel RiPP families including proteusins [START_REF] Freeman | Metagenome mining reveals polytheonamides as posttranslationally modified ribosomal peptides[END_REF] and epipeptides [START_REF] Benjdia | Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis[END_REF] have been shown to contain D-amino acid residues formed by the direct and irreversible epimerization of Lresidues [START_REF] Benjdia | Radical SAM Enzymes and Ribosomally-Synthesized and Posttranslationally Modified Peptides: A Growing Importance in the Microbiomes[END_REF][START_REF] Benjdia | Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis[END_REF][START_REF] Freeman | Metagenome mining reveals polytheonamides as posttranslationally modified ribosomal peptides[END_REF][START_REF] Parent | Mechanistic Investigations of PoyD, a Radical S-Adenosyl-l-methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis[END_REF] . In vivo and in vitro studies have demonstrated that epipeptides induce the expression of LiaRS, a two-component system that orchestrates the cell envelope stress response in Bacillus subtilis [START_REF] Butcher | The yydFGHIJ operon of Bacillus subtilis encodes a peptide that induces the LiaRS two-component system[END_REF][START_REF] Popp | The Epipeptide Biosynthesis Locus epeXEPAB Is Widely Distributed in Firmicutes and Triggers Intrinsic Cell Envelope Stress[END_REF][START_REF] Popp | The Epipeptide YydF Intrinsically Triggers the Cell Envelope Stress Response of Bacillus subtilis and Causes Severe Membrane Perturbations[END_REF] . This system, highly conserved among Firmicutes, regulates the expression of various genes involved in oxidative stress and cell wall antibiotic responses 17- 19 . We have shown that the activity of epipeptides is strictly dependent on the presence of epimerized residues 9 (Figs. 1a,b) and established that they have a distinct mode of action compared to other known antimicrobial peptides [START_REF] Popp | The Epipeptide YydF Intrinsically Triggers the Cell Envelope Stress Response of Bacillus subtilis and Causes Severe Membrane Perturbations[END_REF] . In addition, we have demonstrated that epipeptides are widely distributed in the human microbiota [START_REF] Benjdia | Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis[END_REF][START_REF] Popp | The Epipeptide Biosynthesis Locus epeXEPAB Is Widely Distributed in Firmicutes and Triggers Intrinsic Cell Envelope Stress[END_REF][START_REF] Popp | The Epipeptide YydF Intrinsically Triggers the Cell Envelope Stress Response of Bacillus subtilis and Causes Severe Membrane Perturbations[END_REF] and that epimerizations are catalyzed by a radical S-adenosyl-L-methionine (SAM or AdoMet) enzyme called EpeE [START_REF] Benjdia | Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis[END_REF][START_REF] Popp | The Epipeptide Biosynthesis Locus epeXEPAB Is Widely Distributed in Firmicutes and Triggers Intrinsic Cell Envelope Stress[END_REF] .

Radical SAM enzymes are central biocatalysts in RiPPs biosynthesis and have been shown to install an outstanding diversity of post-translational modifications [START_REF] Balty | Ruminococcin C, an anti-clostridial sactipeptide produced by a prominent member of the human microbiota Ruminococcus gnavus[END_REF][START_REF] Balty | Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes[END_REF][START_REF] Benjdia | Radical SAM Enzymes in the Biosynthesis of Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs)[END_REF][START_REF] Benjdia | The thiostrepton A tryptophan methyltransferase TsrM catalyses a cob(II)alamin-dependent methyl transfer reaction[END_REF][START_REF] Pierre | Thiostrepton tryptophan methyltransferase expands the chemistry of radical SAM enzymes[END_REF][START_REF] Parent | The B12-radical SAM enzyme PoyC catalyzes valine C-beta-methylation during polytheonamide biosynthesis[END_REF][START_REF] Freeman | Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium[END_REF][START_REF] Benjdia | Insights into the catalysis of a lysine-tryptophan bond in bacterial peptides by a SPASM domain radical S-adenosylmethionine (SAM) peptide cyclase[END_REF][25][START_REF] Benjdia | Insights into the catalysis of a lysine-tryptophan bond in bacterial peptides by a SPASM domain radical S-adenosylmethionine (SAM) peptide cyclase[END_REF] . However, until now, no radical SAM enzyme has been crystallized in complex with its peptide substrate properly positioned in the active site, limiting our understanding of their catalysis.

To decipher how radical SAM enzymes introduce post-translational modifications in RiPPs and catalyze epimerization, we have performed EPR, SEC-SAXS and structural analysis of the radical SAM epimerase EpeE from B. subtilis [START_REF] Benjdia | Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis[END_REF][START_REF] Popp | The Epipeptide Biosynthesis Locus epeXEPAB Is Widely Distributed in Firmicutes and Triggers Intrinsic Cell Envelope Stress[END_REF] . We circumvented the intrinsic difficulty to obtain RiPP-enzyme complexes by developing a RiPP-fragment based approach combined with targeted mutagenesis. This novel approach allowed to get at a high resolution, the first crystal structure of a RiPP-modifying radical SAM enzyme in interaction with its substrate. We have also solved the structure of several EpeE mutants and trapped an unexpected adduct on a reactive cysteine residue. Collectively, our work provides a structural and mechanistic rationale for peptide epimerization, a rare case of redox-neutral reaction catalyzed by radical SAM enzymes.

Results

EpeE, a RiPP radical SAM enzyme with a truncated TIM barrel

We crystallized EpeE anoxically in the presence of the S-adenosyl-L-methionine (SAM) or Sadenosylhomocysteine (SAH) cofactor. No significant structural difference was measured (r.m.s.d value of 0.293 Å) however, in subsequent experiments SAH gave high-resolution structures when co-crystallization was performed with peptide substrates. The structure of EpeE with SAH, featuring a homodimer in the asymmetric unit, was determined at resolution of 2.4 Å (Fig. 1c). Out of the 319 protein residues, electron density was observed for residues 1 to 317 in both chains. EpeE exhibits several unique features: first, it is built on a truncated radical SAM domain consisting of an N-terminal partial triose-phosphate isomerase (TIM) barrel fold (i.e. β6/α5) instead of the canonical (β/α)6 TIM barrel. Only the radical SAM enzymes QueE 27 and BtrN [START_REF] Goldman | X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry[END_REF] , which modify small organic substrates, exhibit a shorter core domain (i.e. a β6/α3 and β5/α4 partial TIM barrel, respectively). Second, EpeE is devoid of the α4a helix connecting 4-sheet to α4-helix and found in all structurally characterized RiPPmodifying radical SAM enzymes [START_REF] Davis | Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition[END_REF][START_REF] Grove | Structural Insights into Thioether Bond Formation in the Biosynthesis of Sactipeptides[END_REF] (Extended Data Fig. 1a). Among the radical SAM motifs, we identified the three cysteine residues (C14, C18 and C21) as coordinating the radical SAM [4Fe-4S] cluster with SAH bound, as expected, to the unique iron-atom through its carboxy and amino groups (Fig. 1d). The SAH ligand was further stabilized by the conserved GGE, ribose, "GXIXGXXE" and β6 motifs (Extended Data Fig. 1b) [START_REF] Vey | Structural insights into radical generation by the radical SAM superfamily[END_REF] . However, in the CX 3 CXΦC motif, the Φ residue is not an aromatic residue but H20 which coordinates both N6 and N7 from the adenine moiety and unexpectedly stabilizes the ribose moiety via side-chain H-bond with O2' (Extended Data Fig. 1b-c). Finally, the GGE motif also contains a non-proline Cispeptide bond between the two glycine residues.

A C-terminal domain with an unusual architecture

As a hallmark, radical SAM enzymes involved in peptide or protein post-translational modifications have been shown to possess a C-terminal domain housing additional iron-sulfur clusters. This C-terminal domain is either between 106-115 residues long with two auxiliary iron-sulfur clusters and called a SPASM domain [START_REF] Davis | Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition[END_REF][START_REF] Grove | Structural Insights into Thioether Bond Formation in the Biosynthesis of Sactipeptides[END_REF][START_REF] Haft | Biological systems discovery in silico: radical S-adenosylmethionine protein families and their target peptides for posttranslational modification[END_REF][START_REF] Benjdia | Anaerobic sulfatase-maturating enzyme--a mechanistic link with glycyl radical-activating enzymes?[END_REF] , or significantly shorter and unstructured with a single iron-sulfur cluster and called a twitch domain [START_REF] Grell | Structural and spectroscopic analyses of the sporulation killing factor biosynthetic enzyme SkfB, a bacterial AdoMet radical sactisynthase[END_REF] . Single anomalous diffraction at the peak absorption wavelength of iron was consistent with the presence of four iron-sulfur clusters in the EpeE dimer (Extended Data Table 1). Besides the radical SAM [4Fe-4S] cluster, a single auxiliary cluster (AuxI), best accounted for by a [4Fe-4S] cluster and fully ligated by four cysteine residues (C206, C222, C289 and C292) was identified (Fig. 1d). Two of these 4 cysteine ligands are flanking the conserved -hairpin while the other two are localized in the α4' helix. This iron-sulfur cluster, located at 16.9 Å from the radical SAM cluster, belongs to the C-terminal domain which overall length (111 residues) unexpectedly exceeds the size of a twitch domain. In addition, the β6-β1' bridging loop makes extensive H-bond interactions with the α3' helix and the α3'-α4' loop and extends toward the location where the second cluster is found in SPASM domains (Extended Data Fig. 2) [START_REF] Grell | SPASM and twitch domains in Sadenosylmethionine (SAM) radical enzymes[END_REF] . Additional helices (α3', α4', α5'), absent in twitch domains and involving a tight H-bond network, π-π stacking and hydrophobic interactions, are also present establishing a unique and high secondary structure organization (Extended Data Fig. 1a,2). Overall, the EpeE structure blurs the demarcation between SPASM and twitch domains and defines a transient architecture that we propose to call a T-SPASM domain.

EPR spectroscopic characterization of EpeE

To investigate the properties of the two iron-sulfur clusters, we analyzed EpeE by EPR spectroscopy. The EPR spectrum of EpeE, after reconstitution and reduction, showed a single paramagnetic component characterized by an axial shape with g-values g  = 2.036 and g  = 1.929, which are typical for a reduced S = ½ radical SAM [4Fe-4S] + cluster (Fig. 1e, trace 1) [START_REF] Broderick | Radical S-adenosylmethionine enzymes[END_REF] .

The signal displayed significant relaxation broadening at temperatures above 30 K when recorded under non-saturating microwave power (Extended Data Fig. 3a,b). Consistent with this assignment, it disappeared in the A3-mutant, in which the three cysteines coordinating the radical SAM cluster have been substituted by alanines (Fig. 1e, trace 3). Iron-sulfur determination indicated that, in agreement with its UV-visible spectrum, the reconstituted wildtype EpeE contained 8.6 ±0.5 mol of Fe per mole of protein whereas the A3-mutant contained 4.8 mol ±0.13 of Fe per mole of protein (Extended Data Fig. 3c). This supports the presence of two and one [4Fe-4S] cluster in the wild-type and A3-mutant, respectively. Therefore, the absence of any other signal in the reduced wild-type or A3-mutant is consistent with the fact that the auxiliary cluster is in the EPR silent [4Fe-4S] 2+ redox state, most probably because of its very low redox potential as reported for other radical SAM enzymes [START_REF] Tao | Electron Paramagnetic Resonance Spectroscopic Identification of the Fe-S Clusters in the SPASM Domain-Containing Radical SAM Enzyme PqqE[END_REF][START_REF] Zhu | Structural Properties and Catalytic Implications of the SPASM Domain Iron-Sulfur Clusters in Methylorubrum extorquens PqqE[END_REF] . The addition of SAM to the dithionite-reduced wild-type enzyme induced a substantial change in the resonance position and shape of the EPR spectrum. Notably, a signal from an S = ½ paramagnetic species having all g-values smaller than 2.00 (i.e. g 1 , 2 , 3 = (1.983, 1.871, 1.790)) was detected, as shown in the spectrum measured at 6 K under non-saturating conditions (Fig. 1e, trace 4 & Extended Data Fig. 3a,b). Absent from the A3-mutant (Fig. 1e, trace 6), this signal is assigned to a SAM-bound form of the radical SAM [4Fe-4S] + cluster. Radical SAM [4Fe-4S] + clusters having all g-values smaller than 2.0 have been reported for other enzymes [START_REF] Benjdia | Anaerobic sulfatase-maturating enzyme--a mechanistic link with glycyl radical-activating enzymes?[END_REF][START_REF] Yokoyama | Mechanistic study on the reaction of a radical SAM dehydrogenase BtrN by electron paramagnetic resonance spectroscopy[END_REF][START_REF] Blaszczyk | Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase[END_REF] and generally associated with a cluster form in which SAM binds to the unique iron of cluster, an assignment further supported by HYSCORE measurements (Extended Data Fig. 3d).

Structure of EpeE in complex with a peptide substrate

Initial attempts to crystallize EpeE with its full-length substrate failed, as commonly encountered with RiPP-modifying enzymes. This recurrent failure likely results because of the intrinsically disordered nature of peptides and the fact that many RiPP-modifying enzymes have relaxed substrate specificity, essential to introduce post-translational modifications at distinct sites [START_REF] Weerasinghe | Exploring the Conformational Landscape of a Lanthipeptide Synthetase Using Native Mass Spectrometry[END_REF] . Indeed, EpeE like other radical SAM enzymes [START_REF] Parent | Mechanistic Investigations of PoyD, a Radical S-Adenosyl-l-methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis[END_REF][START_REF] Benjdia | Thioether bond formation by SPASM domain radical SAM enzymes: Calpha H-atom abstraction in subtilosin A biosynthesis[END_REF] introduces multiple posttranslational modifications and must thus adapt to different peptide contexts. To circumvent this inherent difficulty, we designed a peptide library covering the leader and core sequence of the peptide substrate EpeX 1 (Fig. 2a). This RiPP-fragment library was assayed for activity and co-crystallization with EpeE. All peptides containing the core sequence (peptides 4-7) proved to be substrates as shown by labeling experiments performed in deuterated buffer (Extended Data Fig. 4, 5 and Supplementary Tables 12345678) [START_REF] Benjdia | Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis[END_REF][START_REF] Parent | Mechanistic Investigations of PoyD, a Radical S-Adenosyl-l-methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis[END_REF][START_REF] Morinaka | Radical S-adenosyl methionine epimerases: regioselective introduction of diverse D-amino acid patterns into peptide natural products[END_REF] . LC-MS/MS analysis notably confirmed that 2 H-atom incorporation occurred exclusively at the expected epimerization sites. Of note, with peptides 4 and 7, we monitored significant uncoupling between SAM cleavage and epimerization activity, as revealed by 5ʹ-dA isotopic enrichment (Extended Data Fig. 4a,d). In contrast, with peptides 5 and 6, no solvent 2 H-atom was incorporated into 5ʹ-dA, supporting a strict coupling with these substrates (Extended Data Fig. 4b,c & Extended Data Fig. 5).

Remarkably, EpeE co-crystallization assays under anaerobic conditions with SAH and the RiPP-fragment library, led to a high-resolution structure (2.39 Å) of EpeE in complex with peptide 5 (Fig. 2a &b). Crystal form in space group P2 1 2 1 2 1 was identical to the one obtained when crystallizing the substrate-free enzyme, with an asymmetric unit consisting of a homodimer (Extended Data Table 1). Only subtle changes mainly located in the T-SPASM domain could be measured between the free-and bound-substrate structures (r.m.s.d of 0.78 Å, Extended Data Fig. 6). In addition, a strong electron density for the peptide 5 was observed for 7 out of 11 residues (Extended Data Fig. 7a). The peptide fits in the open cavity located at the bottom of the partial TIM barrel and binds between the β1-β5 of the radical SAM domain and the beginning of the T-SPASM domain (Fig. 2b). Peptide 5 is coordinated by a complex network of inter-and intra-molecular interactions folding partially the peptide into a 3 10 -helix (Extended Data Fig. 7b). The residue target of the modification I44 is in the vicinity (3.6 Å) of the aromatic rings of F23 and F228 (Fig. 2c). Interestingly C223, which we proposed as the H-atom donor of the reaction [START_REF] Benjdia | Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis[END_REF] , is part of the hydrophobic pocket around I44 and provides key interactions with I44 including a polar contact via its backbone oxygen and hydrophobic interactions. I44 is also stabilized through its backbone oxygen by hydrogen bonding with the sidechain oxygen of T57 (Fig. 2c). Further stabilization of the substrate is provided by π-π stacking between W43 and the aromatic ring of F175 (EpeE) (π-π centroids 4.6-5.4 Å). Finally, polar interactions with the adenine moiety of SAH (3.28 Å) and hydrophobic contacts with I178 and P225 (3.59 Å) are also involved in stabilizing I44 through W43 (Fig. 2c). The importance of W43 in peptide stabilization is consistent with the fact that the two epimerization sites (I44 and V36), despite lacking sequence homology, are preceded by an aromatic residue. Hence, EpeE appears to recognize a hydrophobic motif for epimerization installation. Further interactions between EpeE and its substrate involve salt bridges between the sidechain of D210 and the sidechains of two substrates residues, R42 and N41, this latter having also a direct interaction with G208 (Fig. 2c). The radical SAM domain through S55 and T57 side chains also contributes to peptide stabilization mainly through polar contacts, while T113 and L84 interact with L45 from the peptide through water contacts. Similarly, N147 (β5) coordinates a water molecule that makes polar contacts with three residues (T43, L45 and R42) from peptide 5 (Fig. 2c). Hence, the structure of the complex revealed that the substrate not only interacts with the two protein domains but also with the SAH cofactor. It also surprisingly showed that the substrate residues preceding I44 adopt a 3 10 -helix secondary structure.

Cys223 as a critical H-atom donor

In the enzyme-substrate complex, the distance between the targeted substrate residue I44 C atom and the C5'-atom of SAM (3.8 Å) is perfectly suited for direct H-atom abstraction from the substrate. Remarkably, on the opposite side of I44, we found C223 (S C223 -C -I44 -C5' SAH angle: 152°) with a sulfur (C223) to C  -atom (I44) distance of 6.3 Å (Fig. 2d). Although this distance is too large for direct interaction between C223 and the transient peptide carboncentered radical formed during catalysis, this distance is likely to shorten due to the loss of I44 stereochemistry. In addition, the structure of the D210A mutant (see below) has revealed that C223 can sample other conformations bringing the sulfhydryl group 2 Å closer to the radical SAM [4Fe-4S] cluster. Altogether, these data are in full agreement with the proposed role of C223 as the H-atom donor of the reaction.

Crystallographic snapshots of an EpeE active-site mutant

Because of the critical role of C223 in the epimerization reaction, we reasoned that the C223A mutant could provide further stabilization to the transient peptide-protein complex. Structure of this mutant perfectly superimposed with the one of the wild-type enzyme (r.m.s.d. value of 0.2945 Å,) with the SAM cofactor in direct interaction with the radical SAM [4Fe-4S] cluster.

EPR analysis of this mutant (Fig. 1e, traces 2 & 5), in the absence or the presence of SAM, showed identical EPR signatures than the wild-type EpeE, supporting that the electronic structure of the radical SAM [4Fe-4S] + cluster is not affected.

Gratifyingly, we solved the structure of the C223A mutant at an atomic resolution with peptide 5 and peptide 6 (1.75 Å and 2.15 Å, respectively) in the presence of SAH. Interactions between peptide 5 and the C223A mutant were overall similar to the wild-type enzyme (r.m.s.d 0.22 Å) (Figs. 3a and Extended Data Fig. 8). However, we obtained electron density for two additional N-terminal amino acid residues located at the very entrance of the active site and forming an additional turn, pointing toward the exit of the active site (Fig. 3a and Extended Data Fig. 8c).

The peptide 6-C223A mutant complex revealed the structure of the full C-terminal end of EpeX 1 (Fig. 3b and Extended Data Fig. 8c). This final peptide segment is stabilized along three sheets from the partial TIM barrel (from  to ) and points toward the exit of the active site with the final peptide residue H49 making polar contacts with I144, S145 and D143. Interestingly, D143 and K171 from EpeE are stacking H49 (Fig. 3b), while in the complex with peptide 5 (lacking H49) and in the substrate-free structure, D143 and K171 are stabilized by a salt bridge in a distinct orientation (Extended Data Fig. 8d,e).

The overlapping sequences of peptides 5 and 6 perfectly superimposed, demonstrating the validity of our RiPP-fragment based approach (Fig. 3c). Notably, in both complexes, the relative location of I44 within the active site, remains the same. Unexpectedly, the presence of H49 provides further inter-chain interactions between the two enzyme subunits (Extended Data Fig. 8d,e). Within the homo-dimeric complex, peptide-peptide interactions are thus likely to significantly contribute to stabilize a productive positioning of the substrate. Exploiting these two atomic-resolution structures, we were able to model, based on the visible electron density, the structure of a peptide spanning 13 residues long (Fig. 3d). In this model, I44 is part of a folded peptide region as a 3 10 -helix while the final GSGH motif has an extended conformation. Remarkably, hydrophobic side-chains are protruding (I44, W43) and alternating with charged residues (E40, R42), reminiscent of the structure of -helical antimicrobial peptides [START_REF] Dathe | Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells[END_REF] .

Structural basis for RiPP epimerization

As shown, in all the peptide-enzyme complexes obtained, I44 is perfectly positioned for C Hatom abstraction (Fig. 4a) and formation of a carbon-centered radical. This transient reaction intermediate is likely to react with the sulfhydryl of C223, leading to epimerization of I44 and the formation of a thiyl radical. With the auxiliary cluster (AuxI) in the vicinity of C223, a consistent function would be as an electron shuttle to the thiyl radical. Only few radical SAM RiPP-modifying enzymes have been spectroscopically characterized [START_REF] Tao | Electron Paramagnetic Resonance Spectroscopic Identification of the Fe-S Clusters in the SPASM Domain-Containing Radical SAM Enzyme PqqE[END_REF][START_REF] Ayikpoe | Spectroscopic and Electrochemical Characterization of the Mycofactocin Biosynthetic Protein, MftC, Provides Insight into Its Redox Flipping Mechanism[END_REF][START_REF] Balo | Trapping a cross-linked lysine-tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB[END_REF] but for some of them, the auxiliary cluster (AuxI) proved to be a low-potential [4Fe-4S] cluster [START_REF] Tao | Electron Paramagnetic Resonance Spectroscopic Identification of the Fe-S Clusters in the SPASM Domain-Containing Radical SAM Enzyme PqqE[END_REF] . In addition, recent investigations have shown that substrate binding can tune the redox properties of the AuxI cluster, further supporting a role as an electron shuttle [START_REF] Balo | Trapping a cross-linked lysine-tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB[END_REF] . In EpeE, AuxI is likely to fulfill a similar function with two tyrosine residues (Y209 and Y2) located beneath the cluster and stabilized by main chain interactions, hydrogen bonds and π-π stacking, ideally positioned to serve as an electron conduit from the protein surface (Fig. 4a). In addition, we also identified in the immediate environment of C223, only one titratable residue: D210. This residue provides key electrostatic interactions to the substrate (Figs. 2c, 3a and 3b) and is stabilized by a polar bond with T5 (Extended Data Fig. 9a).

To probe for the function of these residues, we generated several mutants by substituting the two tyrosines with phenylalanine residues (Y2F-Y209F mutant) and by replacing D210 with an alanine residue (D210A mutant). The activity of these mutants was assayed using peptide 6 as substrate and a physiological reducing system (NADPH/flavodoxin/flavodoxin reductase).

As shown, we obtained a tight coupling between SAM cleavage (5'-dA production) and peptide epimerization with the wild-type enzyme (Fig. 4b). In contrast, with the C223A mutant, epimerization was strongly impaired while SAM cleavage activity remained similar to the wildtype enzyme. Hence, with the C223A mutant, only the epimerization reaction is affected. The two other mutants Y2F-Y209F and D210A had similar activities compared to the wild-type EpeE (Fig. 4b). However, under in vitro conditions, the physiological roles of these amino acid residues might be difficult to evidence because of the excess of reductant and electrons present in the reaction mixtures.

The high-resolution structure of the D210A mutant in complex with SAH (1.95 Å) was similar to the wild-type enzyme (r.m.s.d. of 0.25 Å). However, it exhibited two major differences: first, C223 adopted a distinct orientation bringing the sulfhydryl group 2 Å closer toward the SAH cofactor. Second, we noted the presence on C223 of an additional electron density (Extended Data Fig. 9b). This extra electron density was modeled, after analysis of the 2F o -F c map maximum intensity, as a second sulfhydryl group (Extended Data Fig. 9b). The presence of a persulfur on a reactive cysteine has been reported in several systems including cytochrome thiosulfate dehydrogenase [START_REF] Brito | Thiosulfate dehydrogenase (TsdA) from Allochromatium vinosum: structural and functional insights into thiosulfate oxidation[END_REF] or the cysteine desulfurase NifS [START_REF] Nakamura | Snapshots of PLP-substrate and PLP-product external aldimines as intermediates in two types of cysteine desulfurase enzymes[END_REF] . In the D210A mutant, the origin of this additional sulfur atom is unknown but it supports that C223 is a reactive residue. Careful analysis of all structures showed that this adduct and this sidechain orientation were only present in the D210A mutant, supporting that D210 plays an important role to stabilize C223. Finally, polar contacts between D210 and T5 sidechains are also reminiscent of the catalytic "acid-alcohol pair" found in cytochrome P450 [START_REF] Denisov | Structure and chemistry of cytochrome P450[END_REF] and involved in proton transfer within the active site. Collectively, these data support that D210 interacts with C223 and could play an important role not only in substrate stabilization and positioning, but also for C223 reactivity.

Discussion

Despite major advances in our understanding of RiPP biosynthesis, core peptide recognition by RiPP enzyme active sites is largely unknown. These enzymes must often recognize multiple modification sites to generate their final products, supporting a relaxed substrate specificity.

Recent studies have shown that RiPP-modifying enzymes including radical SAM enzymes, while catalyzing a vast array of chemical transformations [START_REF] Montalban-Lopez | New developments in RiPP discovery, enzymology and engineering[END_REF][START_REF] Benjdia | Radical SAM Enzymes and Ribosomally-Synthesized and Posttranslationally Modified Peptides: A Growing Importance in the Microbiomes[END_REF] , share conserved structural features such as an RRE to interact with the leader peptide [START_REF] Burkhart | A prevalent peptide-binding domain guides ribosomal natural product biosynthesis[END_REF] and ensure the fidelity of the post-translational modifications. However, this structural element is absent from many enzymes questioning the processes underpinning their specificity.

To address these fundamental questions, we have investigated the radical SAM epimerase EpeE 9 alone and in complex with several peptide substrates. Structural and EPR analyses demonstrate that EpeE contains two [4Fe-4S] clusters and has an unusual architecture. Its compact fold markedly differs from all known RiPP-modifying radical SAM enzymes by the notable absence of an RRE domain and of the α6 and α4a helices from the partial TIM barrel.

In addition, EpeE possesses a unique C-terminal domain which shares the single [4Fe-4S] cluster of twitch-domains but with an overall fold reminiscent of the SPASM-domain, that we propose to call a T-SPASM domain (Figs. 1 &2).

By generating a RiPP-fragment library, we were able to obtain high-resolution crystallographic snapshots of a RiPP-modifying radical SAM enzyme in a pre-catalytic state (Figs. 2b,c and Extended Data Fig. 8) with its substrate properly located in the active site. Currently, only two structures of radical SAM enzymes catalyzing RiPP post-translational modifications have been solved in complex with peptide substrates [START_REF] Davis | Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition[END_REF][START_REF] Grove | Structural Insights into Thioether Bond Formation in the Biosynthesis of Sactipeptides[END_REF] . Intriguingly, in SuiB, which catalyzes the formation of carbon-carbon bonds, the leader peptide was found in the partial TIM barrel instead of the RRE and no electron density was present for the core peptide. For CteB which catalyzes thioether bond formation, only the leader peptide sequence and the first three residues of the core region were visible in the co-crystallized structure [START_REF] Grove | Structural Insights into Thioether Bond Formation in the Biosynthesis of Sactipeptides[END_REF] .

In sharp contrast, the structures of EpeE and its C223A mutant in complex with different peptide substrates revealed that I44, the amino acid residue target of the modification, is in an ideal position for direct C α H-atom abstraction by the 5ʹ-dA radical (I44 C  -atom-C5' distance of 3.8 Å). Structural analysis also revealed that a conserved cysteine residue (C223) is located beneath I44 to serve as an H-atom donor. By using a physiological reductant system, we demonstrated that while the SAM cleavage activity is not affected by mutation of C223, the second part of the reaction (i.e. H-atom transfer to the carbon-centered radical) is hampered leading to a strong uncoupling between SAM cleavage and peptide epimerization (Fig. 4b). Interestingly, while the distance in the peptide-enzyme complex is too long (6.3 Å, Fig. 2d) for a direct interaction between C223 and the C  -atom of I44, structural analysis of the D210A mutant revealed that C223 can sample various conformations, bringing the sulfur atom closer to the I44 C  -atom. Altogether, these results support that radical SAM peptide epimerases effectively use a cysteine residue as critical H-atom donor, a strategy likely common to all radical SAM epimerases [START_REF] Parent | Mechanistic Investigations of PoyD, a Radical S-Adenosyl-l-methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis[END_REF][START_REF] Kudo | Characterization of a radical Sadenosyl-L-methionine epimerase, NeoN, in the last step of neomycin B biosynthesis[END_REF][START_REF] Besandre | HygY Is a Twitch Radical SAM Epimerase with Latent Dehydrogenase Activity Revealed upon Mutation of a Single Cysteine Residue[END_REF] .

Another advantage of our RiPP-fragment based approach was to gain structural information regarding RiPP structure during catalysis. Indeed, based on the structure of several peptideenzyme complexes, we were able to model up to 13 substrate residues, starting from the residue after V36 up to the C-terminal end of EpeX. This model reveals that EpeX is partially folded as a 3 10 -helix in the enzyme active site with protruding hydrophobic side-chains. Although 3 10 -helices have been identified in other RiPPs, the core region is usually unstructured or with a U-turn shape in RiPP-enzyme complexes [START_REF] Dong | Mechanistic Basis for Ribosomal Peptide Backbone Modifications[END_REF][START_REF] Zhao | Structural Basis for a Dual Function ATP Grasp Ligase That Installs Single and Bicyclic omega-Ester Macrocycles in a New Multicore RiPP Natural Product[END_REF][START_REF] Song | Molecular mechanism underlying substrate recognition of the peptide macrocyclase PsnB[END_REF][START_REF] Miller | Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis[END_REF] .

In sharp contrast to the known structures of (non-radical SAM) RiPP-modifying enzymes [START_REF] Dong | Mechanistic Basis for Ribosomal Peptide Backbone Modifications[END_REF][START_REF] Zhao | Structural Basis for a Dual Function ATP Grasp Ligase That Installs Single and Bicyclic omega-Ester Macrocycles in a New Multicore RiPP Natural Product[END_REF][START_REF] Song | Molecular mechanism underlying substrate recognition of the peptide macrocyclase PsnB[END_REF][START_REF] Miller | Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis[END_REF] , only minor rearrangements occurred in EpeE upon substrate binding. Peptide-enzyme interactions are driven not only by residues from the radical SAM domain but also by residues from the T-SPASM domain and the SAM cofactor itself, forging a complex network of interactions that ensure substrate recognition in the absence of RRE. Interestingly, a similar strategy implying interactions between the substrate backbone with the protein domains and the SAM cofactor, has been recently reported for the B 12 -dependent radical SAM enzyme Mmp10 [START_REF] Fyfe | Crystallographic snapshots of a B12-dependent radical SAM methyltransferase[END_REF] .

Another intriguing finding of this study is that with peptide 6, not only the full C-terminal end of epipeptide could be accommodated in the EpeE dimer without steric clashes but unexpectedly, further stabilization was provided by the last residue. Indeed, H49, stacked between K171 and D143, directly interacts through inter-chain interactions with H49 from the second subunit (Extended Data Fig. 8d,e), supporting a physiological role for the dimer (Fig. 5a). To validate this model, we performed anaerobic SEC-SAXS analysis in order to determine the oligomerization state of EpeE in solution. SEC-SAXS analysis showed that anaerobically reconstituted EpeE in the presence of SAM behaved as a homogenous globular and folded protein (Fig. 5b) with a radius of gyration of 28.7 Å and an estimated molecular weight of 73-71 kDa. These experimental data are consistent with EpeE being exclusively a dimer in solution and are in full agreement with our structural analysis. Additionally, surface interaction calculations using the PDBePISA server 57 also supported that the dimer is the most stable species (Supplementary Tables 910). This dimer provides an elegant structural rationale for the installation of the two epimerizations in epipeptides. Initially, two peptide molecules interact within the EpeE dimer, positioning I44 in the active site for direct attack by the 5'-dA radical.

The presence of a hydrophobic residue before each targeted residue is also likely to provide key stabilization. Following H-atom abstraction, the amino acid configuration is lost (Fig. 5c) and the radical substrate intermediate is quenched by C223, which is ideally located on the opposite side of the targeted residue. Similar critical cysteine residues have been identified in all radical SAM epimerases investigated to date [START_REF] Parent | Mechanistic Investigations of PoyD, a Radical S-Adenosyl-l-methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis[END_REF][START_REF] Kudo | Characterization of a radical Sadenosyl-L-methionine epimerase, NeoN, in the last step of neomycin B biosynthesis[END_REF][START_REF] Besandre | HygY Is a Twitch Radical SAM Epimerase with Latent Dehydrogenase Activity Revealed upon Mutation of a Single Cysteine Residue[END_REF] . After configuration inversion, a transient thiyl radical is generated on C223. D210 which forms a pair with T5 and stabilizes C223, is the only residue that could serve to regenerate the thiyl radical using a proton-coupled electron transfer pathway. Following, the first epimerization event, the dimer progresses toward V36 which is already in the vicinity the active site (Fig. 5a). In support of this model, not only we have always crystallized peptides with I44 positioned for H-atom abstraction but also I44 was always the hotspot for epimerization (Extended Data Fig. 4), suggesting a C-to-N directionality in EpeE like in PoyD [START_REF] Parent | Mechanistic Investigations of PoyD, a Radical S-Adenosyl-l-methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis[END_REF] .

In conclusion, our study reports an unprecedented binding mode for a RiPP-modifying enzyme and expands the structural diversity of radical SAM enzymes catalyzing RiPP post-translational modifications. Unexpectedly, the EpeE structure reveals that inter-chain interactions could play a significant role for the sequential installation of epimerizations. Finally, this work illuminates how radical SAM enzymes catalyze peptide epimerization, a rare example of redox-neutral reaction, delineating novel reactivity in enzyme active sites.

Figure legends

Fig. 1 -Epipeptide biosynthesis and overall structure of the EpeE radical SAM epimerase. a, Sequence of EpeX precursor with the leader (white circles) and the core peptide sequence (blue circles) indicated. After modification by EpeE, the mature epipeptide is produced with epimerization of V36 and I44 (in red). b, Epimerization reaction catalyzed by EpeE showing the C α H-atom, target of the modification, in red. c, Overall structure of the EpeE homodimer in the asymmetric unit (left panel). Radical SAM domain (light blue, residues 1-175) and T-SPASM domain coordinating the auxiliary (AuxI) [4Fe-4S] cluster (teal, residues 206-317). d, Crystal structure of EpeE with bound SAH. The [4Fe-4S] and AuxI clusters are shown as balls and sticks (yellow and orange spheres) and SAH as sticks (green). The omit map (blue mesh) of SAH and [4Fe-4S] clusters are contoured at 3σ. AuxI: auxiliary cluster. e, EPR analysis of EpeE. X-band CW EPR spectra of dithionite-reduced reconstituted samples of EpeE WT (traces 1, 4), C223A mutant (traces 2, 5) and A3-mutant (traces 3, 6) and in the absence (traces 1-3) and presence (traces 4-6) of an excess of SAM. Experimental conditions: temperature, 15 K (traces 1-3) or 6 K (trace 4-6), microwave power, 0.1 mW, field modulation amplitude, 1 mT, microwave frequency, 9.48 GHz, number of accumulations, 8 (1-3), or 16 (4-6). Protein concentration, 320 µM (1, 4), 223 µM (2, 5), 380 µM (3, 6), SAM concentration, 1 mM. 10). c, Proposed mechanism for EpeE. Following the reduction of the radical SAM cluster, the 5'-dA radical generated, abstracts the C  H-atom of the target residue. The carbon-centered radical formed on the substrate reacts with C223, leading to configuration inversion of I44 and formation of a thiyl radical on C223. The AuxI [4Fe-4S] cluster, likely assisted by D210, is ideally positioned to regenerate C223 through a protoncoupled electron transfer pathway.

Methods

Cloning of EpeE D210A and Y2F-Y209F mutants

The EpeE tyrosine mutants were obtained by site-directed mutagenesis with the plasmid pET28-Strep-TEV-EpeE used as DNA template. To generate the Y2F-Y209F mutant the following primers were used: 5'-CAT ATG TTC AAC AAA ACC GTG AGC-3' ; 5'-TCC GGG TTT CGA TAT TGT GTA TCA TC-3' ; 5'-ACA ATA TCG AAA CCC GGA CAA TGC AG-3'. PCR reactions typically contained 1 μM of each primer, 250 μM of each dNTP, 1 μl of High Fidelity Polymerase (Thermo), ~50 ng DNA template and performed using the following PCR cycling parameters: 1 cycle at 95°C for 2 min followed by 36 cycles of 95°C for 30 sec (denaturation), 55-60°C for 30 sec (hybridation), 72°C for 90 s (extension) and 1 cycle at 72°C for 10 min. PCR products were purified on 1-2% agarose gel, digested with NdeI and XhoI (NEB), purified and ligated into NdeI/XhoI digested pET28-Strep-TEV plasmid. The D210A mutant gene was synthesis by GeneCust and ligated in plasmid pET28. The ligation products were transformed into chemically competent E. coli BL21 (DE3) star strain. Clones were selected on LB agar plate containing 50 μg.mL -1 kanamycine. One clone was selected after we checked the sequence of the targeted gene by DNA sequencing.

Protein purification

The constructs of wild-type and C223A mutant streptavidin-tagged EpeE were the same as previously published [START_REF] Benjdia | Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis[END_REF] . All recombinant proteins were overexpressed and purified as follows. Typically, 9 L of E. coli BL21 cells transformed with pET28 plasmid containing the construct were grown in Luria-Broth (LB) media at 37°C with 50 µg/mL Kanamycin, and protein expression induced at Abs600nm ~ 0.6 with 700 µM IPTG. Overexpression was conducted overnight and cells resuspended in Buffer A (500 mM NaCl, 50 mM TrisHCl, pH 8.0) containing 0.5% Triton X-100, protease inhibitors and sonicated for 4 min on ice. Cell debris were pelleted by ultracentrifugation at 45,000 g (Beckman coulter) before loading supernatant on Streptactin resin (IBA) equilibrated in buffer A. After washing in buffer A, the recombinant protein was eluted with buffer A supplemented by 3 mM desthiobiotin. Protein was aliquoted, flash frozen in liquid N2 and stored at -80°C. Concentration was measured by absorbance at 280 (using ProtParam calculated ε and molecular weights) and purity assessed by SDS-PAGE on 12% gels and PageBlue staining.

Iron sulfur clusters reconstitution

In vitro reconstitution of the EpeE [4Fe-4S] clusters was achieved in presence of 3 mM DTT by addition of 8 molar excess of (NH4)2Fe(SO4)2 and Na2S. After incubation under anaerobic atmosphere, excess of unbound iron and sulfur was removed onto a desalting column against buffer A with 3 mM DTT. Reconstitution of the protein was verified by recording UV-visible spectra (250-700 nm).

Peptide synthesis

The EpeX peptide 1 and RiPP-fragments peptide 2 (KKEITNNETVKNLEFKGLLDESQKLAKVNDL), peptide 3 (KNLEFKGLLDESQKLAKVNDL), peptide 4 (WYFVKSKEN), peptide 5 (FVKSKENRWIL), peptide 6 (KENRWILGSGH), peptide 7 (NRWILGSGH) were synthesized by solid-phase synthesis (ProteoGenix, France). In bold, residues targeted by EpeE.

EPR analysis

EPR samples were prepared immediately after reconstitution of the iron-sulfur clusters in the glove box. Reduction was achieved in 5 minutes by the addition of 3 mM sodium dithionite. Samples in the presence of SAM were prepared by addition of 3-5 fold stoichiometric excess of SAM. Samples were then frozen in liquid isopentane in the glove box and then maintained in liquid nitrogen until EPR measurements were performed. EPR spectra were recorded on a Bruker ElexSys-500 X-band spectrometer equipped with a standard rectangular cavity (ST4102) fitted to an Oxford Instruments liquid helium cryostat (ESR900) and temperature control system. The spectrometer settings were as follows: microwave frequency, 9.48 GHz, conversion time of 80 ms, modulation amplitude of 1 mT, and modulation frequency of 100 kHz. Other settings are given in the corresponding figure captions. HYSCORE experiments were measured at 6 K using a Bruker EleXsys E580 spectrometer equipped with an ER4118X-MD5 dielectric resonator and an Oxford Instruments CF 935 cryostat. This four-pulse experiment (/2--/2-t1--t2-/2--echo) was employed with an appropriate 8 step phase-cycling scheme to eliminate unwanted features from the experimental electron spin echo envelopes. The intensity of the echo after the fourth pulse was integrated over 32 ns with varied t2 and t1 and constant .

The length of a π/2 pulse was 12 ns and of a π pulse 24 ns. A  value of 132 ns and a shot repetition rate of 1 kHz were used. HYSCORE data were collected in the form of 2D time-domain patterns containing 256  256 points with steps of 16 ns. Spectra were recorded at the magnetic field value corresponding to the maximum absorption of the [4Fe-4S] 1+ signal in the absence of SAM (corresponding to g = 1.929). HYSCORE spectra were processed using Bruker's Xepr software. Relaxation decays were subtracted (fitting by 3 rd order polynomial functions) followed by zero-filling to 1024 points and tapering with a Hamming window, before 2D Fourier transformation which finally gives the spectrum in frequency domain. Processed data were then imported into Matlab (The MathWorks Inc., Natick, MA) for plotting. HYSCORE spectra are shown in absolute value mode and are presented as contour plots.

Enzyme assay

The activity of EpeE towards the synthetic peptides was assayed in deuterated buffer (150 mM KCl, 25 mM TrisDCl, pH 8.0, DTT 3 mM); all compounds were dissolved in D2O and the reconstituted protein was buffer exchanged against deuterated buffer. EpeE (235 µM) was incubated with 3 mM DTT, 3 mM SAM and 2.25 mM peptide. The reactions were initiated by addition of 3 mM sodium dithionite.

Rg (Å)

Rg (Å)

Figure or Table #

  Figure or Table#

  Please group Extended Data items by type, in sequential order. Total number of items (Figs. + Tables) must not exceed 10.

Figure

  Figure/Table title

  Figure/Table Legend

  If you are citing a reference for the first time in these legends, please include all new references in the main text Methods References section, and carry on the numbering from the main References section of the paper. If your paper does not have a Methods section, include all new references at the end of the main Reference list.

7

 7

  The peptide 5 was built for 7 out of 11 residues (KENRWIL) according to the electron density. The omit map (blue mesh) of peptide 5 (in pink sticks) is contoured at 3σ. SAH is depicted in stick, the radical SAM [4Fe-4S] and AuxI clusters are shown as spheres. b, Peptide 5 fold. The peptide is shown in salmon (chain C) and orange (chain D) and colored by atom type. Intramolecular interactions are depicted in black dashed line.

Fig. 2 - 2 , 3 , 4 , 5 , 6 & 7 .

 2234567 Fig. 2 -Structure of EpeE in interaction with a peptide substrate and SAH cofactor. a, Sequences of the peptide substrates assayed: EpeX (peptide 1) and the RiPP-fragments: peptides 2, 3, 4, 5, 6 & 7. The leader peptide is shown in white circles and the core peptide is indicated by blue circles. The two residues, targets of the modification by EpeE (i.e. V36 and I44) are shown in red in blue circles. b, Overall structure of EpeE in complex with the peptide 5 (left panel). The bound peptide 5 (residues K 39 ENRWIL 45 ) is depicted in pink sticks, SAH is shown in green and the two [4Fe-4S] clusters are in yellow and orange. APBS-derived surface electrostatics of EpeE reveals three distinct electrostatic potentials in the EpeE peptide binding pocket (right panel). Surface colored according to electrostatic potential with positively charged regions in blue and negatively charged regions in red. The bound peptide is shown in pink sticks. c, Coordination of the peptide 5 (pink sticks) by both EpeE domains. The binding pocket within 5 Å of the peptide consists of 6 hydrophobic residues (F23, F175, F228, L84, I85, I178), five polar uncharged residues (S55, T57, T83, T113, S224), and three charged residues (N9, N147, D210), in addition to C223, P176 and P225. SAH, the radical SAM [4Fe-4S] and AuxI clusters are shown as sticks. Amino acid residues in light blue belong to the radical SAM domain and the ones in teal are from the T-SPASM domain. d, Close-up of the EpeE active site showing SAH in green, the peptide 5 in pink, C223 in teal and the two [4Fe-4S] clusters in yellow and orange. The distances between the C5′ atom of SAH and the C  atom of I44 from peptide 5 (3.8 Å) and between I44 and the sulfur-atom of C223 from EpeE (6.3 Å) are indicated by dashed lines.

Fig. 3 -Fig. 4 -

 34 Fig. 3 -Structures of C223A mutant in interaction with two peptide substrates and SAH cofactor. a, Close-up of EpeE C223A mutant active site showing protein residues from the radical SAM (in light blue) and the T-SPASM (in teal) domains in interaction with the peptide 5 (in pink). Dashed lines show polar contacts between peptide 5 and protein residues. SAH is depicted in green sticks and both [4Fe-4S] clusters are shown in yellow and orange spheres. b, Close-up of EpeE C223A mutant active site showing protein residues from the radical SAM (in light blue) and the T-SPASM (in teal) domains in interactions with the peptide 6 (in light

Fig. 5 -

 5 Fig.5-Substrate interaction and proposed mechanism for EpeE. a, Model of epimerization catalyzed by EpeE on the EpeX peptide. In the EpeE dimer, two peptide molecules (EpeX) interact through inter-(H49) and intra-chain interactions with I44 positioned in the active site for an attack by the 5'-dA radical. After the first epimerization event, the dimer progresses toward V36, the second epimerized residue. b, SEC-SAXS analysis of EpeE in the presence of SAM (panel 1) or SAM and peptide 6 (panel 2). The UV visible signal at λ = 280 nm (blue) and SAXS intensity at zero angle (red) is superimposed with the computed Rg from SAXS data (open circles). SAXS curves of EpeE in the presence of SAM (panel 3) and EpeE in the presence of SAM and peptide 6 (panel 4). Fit curves using PepsiSAXS are indicated by a red trace and  2 value determined between the experimental data and the simulated model (see Supplementary Table10). c, Proposed mechanism for EpeE. Following the reduction of the radical SAM cluster, the 5'-dA radical generated, abstracts the C  H-atom of the target residue. The carbon-centered radical formed on the substrate reacts with C223, leading to configuration inversion of I44 and formation of a thiyl radical on C223. The AuxI [4Fe-4S] cluster, likely assisted by D210, is ideally positioned to regenerate C223 through a protoncoupled electron transfer pathway.

4, 5, 6 and 7 and the reaction products formed after

	incubation	with	spectrum of peptide 6 (upper panel) and the
	EpeE.		epimerized peptide product (lower panel). (see
			Supplementary Tables 5
		ED_Figure5.jp
		g

a, Mass fragmentation spectrum of peptide 4 (upper panel) and the epimerized peptide product (lower panel). (see Supplementary Tables 1 -2 for full assignment). b, Mass fragmentation spectrum of peptide 5 (upper panel) and the epimerized peptide product (lower panel). (see Supplementary Tables 3 -4 for full assignment). c, Mass fragmentation

-6 for full assignment). d,

  Mass fragmentation spectrum of peptide 7 (upper panel) and the epimerized peptide product (lower panel). (see Supplementary

Tables 7 -8 for full assignment).

  The relevant ions with a mass shift of +1 Da due to 2 H incorporation after reaction with EpeE are highlighted.

Table . 1 Crystallographic data and refinement statistics Table_1.pdf 1. Supplementary Information: A. PDF Files

 .

	Reporting Summary	Yes	nr-reporting-
			summary.pdf
	Peer Review	No	OFFICE USE
	Information		ONLY
	2. Source Data
	Parent Figure or	Filename		Data description
	Table	Whole original file name including	i.e.: Unprocessed western Blots and/or gels, Statistical
		extension. i.e.:		Source Data, etc.
		Smith_SourceData_Fig1.xls, or
		Smith_
		Unmodified_Gels_Fig1.pdf
	Source Data Fig. 4 Source_data_file.csv	Statistical source data for Fig. 4b
	Item	Present?	Filename	A brief, numerical description of file
			Whole original file	contents.
			name including	i.e.: Supplementary Figures 1-4, Supplementary
			extension. i.e.:	Discussion, and Supplementary Tables 1-4.
			Smith_SI.pdf. The
			extension must be
			.pdf

Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme Xavier Kubiak 1+ , Ivan Polsinelli 1+ , Leonard M. G. Chavas 3+ , Cameron D Fyfe 1+ , Alain Guillot 1 , Laura Fradale 1 , Clemence Brewee 1 , Stéphane Grimaldi 4 , Guillaume Gerbaud 4 , Aurélien Thureau 2 , Pierre Legrand 2 , Olivier Berteau 1 * & Alhosna Benjdia 1 *

	1 Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, ChemSyBio, 78350
	Jouy-en-Josas, France
	2 Synchrotron SOLEIL, HelioBio group, L'Orme des Merisiers, 91190 Gif sur-Yvette,
	France
	3 Nagoya University, Nagoya 464-8603, Japan.
	4 Aix Marseille Univ, CNRS, BIP, IM2B, IMM, Marseille, France
	*Corresponding authors. Email: Olivier.Berteau@inrae.fr; Alhosna.Benjdia@inrae.fr
	+ Equal contribution

D-

amino acid residues have been reported in countless peptides and natural products including ribosomally-synthesized and post-translationally modified peptides (RiPPs), where they are critical for the bioactivity of several antibiotics and toxins. Recently, radical S-adenosyl-L-methionine (SAM) enzymes have emerged as the only biocatalysts capable of installing direct and irreversible epimerization in RiPPs. However, the mechanism underpinning this biochemical process is ill-understood and the structural basis for this post-translational modification remains unknown. Here we report an atomic-resolution crystal structure of a radical SAM RiPP-modifying enzyme in complex with its substrate properly positioned in the active site. Crystallographic snapshots, SEC-SAXS, EPR spectroscopy and biochemical analyses revealed how epimerizations are installed in RiPPs and support a unprecedented enzyme mechanism for peptide epimerization. Collectively, our study brings unique perspectives on how radical SAM enzymes interact with RiPPs and catalyze post-translational modifications in natural products.

Acknowledgments

This work was supported by the French National Research Agency (ANR grants: ANR-17-CE11-0014 to O.B.; ANR-20-CE44-0005 to O.B.). The authors are grateful to the EPR facilities available at the French EPR network (IR CNRS 3443, now INFRANALYTICS, FR2054) and the Aix-Marseille University EPR center. We acknowledge SOLEIL (Saint-Aubin, France) for provision of synchrotron radiation facilities and we would like to thank the PROXIMA-1 and SWING staff for assistance in using the beamline.

Data Availability

Atomic coordinates and structure factors for the reported crystal structures in this work have been deposited in the Protein Data Bank upon accession codes: 8AI1, 8AI2, 8AI3, 8AI4, 8AI5 & 8AI6. SAXS data have been deposited in the Small Angle Scattering Biological Data Bank upon accession codes SASDRS7, SASDRR7. The data for this study are available within the paper and its Supplementary Information. Source data are provided with this paper.

Competing Interests Statement

The authors declare no competing interests.

Additional information

Extended data is available for this paper.

For the kinetic experiments, the EpeE WT, C223A, D210A and Y2F-Y209F mutants (100µM) were incubated with 1 mM SAM, 500µM peptide 6 and reactions were initiated with the addition of flavodoxin/flavodoxin reductase and 2.7 mM NADPH.

All reactions were performed at 25°C and 15 µL aliquots sampled overtime for LC-MS/MS analysis.

Liquid chromatography-mass spectrometry/mass spectrometry analysis

LC-MS/MS analysis were achieved using a Q-Exactive Focus mass spectrometer (ThermoFisher Scientific) with the HESI2 electrospray ion source associated to a Vanquish Flex LC system (Thermo Fisher Scientific). Molecule separation was performed on a Zorbax Eclipse Plus C18 column (2.1X50mm, 1.8µm, RRHT, Agilent). Samples were dilute one hundred time before injection with TFA 0.1% or in acetonitrile 50%, to discard the protein by precipitation. Peptides 4, 5, 6 or 7 were eluted on the reverse phase column by an acetonitrile gradient with formic acid 0.1% as ion pairing agent and detected by mass spectrometry in positive mode. The doubly charged ions corresponding to the peptides 4, 5, 6, 7 were selected for HCD fragmentation step at 35 % or 20 % of NCE. For detection and quantitation of the epimerized peptide 5 by mass spectrometry, the samples were hydrolyzed using trypsin Promega (V5111) to produce the peptide fragment WILGSGH and extract the corresponding MS signal of the doubly charge ion (385.21 2+ ). This method enables us to quantify the epimerized and unmodified peptides. Unmodified peptide fragment eluted at 13.7 min and the epimerized fragment at 18.2 min from the column. The 5ʹ-dA produced during in vitro reactions with EpeE (wild-type and D210A, C223A and Y2F-Y209F mutants) was quantified in the sample by mass spectrometry using a direct standard curve of 5ʹ-dA.

Crystallization of EpeE, EpeE C223A, EpeE D210A mutants and complexes with peptides 5 and 6

For crystallization purposes, the freshly reconstituted protein was further purified by size exclusion on a Superdex-200 10/300 GL Increase column (Äkta system, GE Healthcare) to achieve homogeneity in the protein preparation. M Tris-HCl pH 8.5, 14-20% PEG 1,000) with protein, SAH (0.5 mM) and peptide (5 mM). All crystals were typically obtained within 24 hours under anaerobic atmosphere and flash cooled in liquid N2 under anaerobic conditions.

Crystallographic structure determinations

X-ray diffraction data were recorded on PROXIMA-1 beamline at Synchrotron SOLEIL [START_REF] Coati | Status of the crystallography beamlines at synchrotron SOLEIL⋆[END_REF] , equipped with a Pilatus-6M hybrid pixel detector [START_REF] Kraft | Performance of single-photon-counting PILATUS detector modules[END_REF] and a three-axis SmarGon goniometer. An X-ray energy of 7.14 keV was chosen for the single anomalous phasing experiments, for which two sets of images were recorded at two different Chi angles to favor obtaining a more complete merged data at 2.99 Å resolution. The X-ray energy was 12.67 keV for subsequent data collection. For the experimental phasing measurements, the X-ray energy was attenuated to 5% of the beam intensity, providing with a flux of 1.0 x 10 10 photons.s -1 at the sample position; the more standard data at 12.67 keV were recorded with 20% of the beam intensity. Phasing and data analysis and processing was performed using SHARP/AutoSHARP (SHARP 2.8.12 and Sushi 3.10.11), and the CCP4 suite (7.1.016). Data were processed with XDS 2022/01/10 60 through the xdsme scripting package [START_REF] Legrand | XDSME: XDS Made Easier[END_REF] . The ( hkl ) and ( -h-k-l ) reflections were treated separately in all steps, including scaling and merging. Data sets were scaled with XSCALE 2022/01/10 60 , converted to MTZ format with POINTLESS 1.12.13 [START_REF] Evans | Scaling and assessment of data quality[END_REF] and merged with AIMLESS 0.7.8 [START_REF] Evans | How good are my data and what is the resolution?[END_REF] , as implemented within the xdsme automated procedure.

For the EpeE phasing dataset, structure-factor amplitudes were obtained with TRUNCATE 8.0.010 [START_REF] Winn | Overview of the CCP 4 suite and current developments[END_REF] . The Matthews coefficient suggested that the crystal contained two monomers per asymmetric unit. Four iron cluster sites were located with SHELXC/D 2016/1 [START_REF] Sheldrick | A short history of SHELX[END_REF] , and their respective positions were refined with SHELXE 2019/1 (through HKL2MAP) [START_REF] Pape | 2MAP : a graphical user interface for macromolecular phasing with SHELX programs[END_REF] and Phaser 2.8 [START_REF] Mccoy | Phaser crystallographic software[END_REF] . Initial correct substructures were determined immediately with SHELXD 2013/2, with two well-separated clusters in the plot of CCall versus CCweak. The phase information was improved using the existing non-crystallographic symmetry (NCS) between the two molecules within the asymmetric unit. One helix surrounding the iron clusters was docked in the original electron density using

Coot [START_REF] Emsley | Features and development of Coot[END_REF] to facilitate the calculation of the matrix. Given this matrix, Phaser unequivocally identified the rotation and translation parameters of two monomers and confirmed the space group P212121. Side chains were placed by automated model building with Buccaneer 1.6.12/REFMAC5 69 . Missing residues, water molecules and ligands were fitted in the 2Fo-Fc and Fo-Fc electron-density maps using Coot. For structure refinements of the subsequent models, phases were introduced from the original structure as a template coordinates (Phaser) and the structures were refined iteratively with REFMAC5 or BUSTER (version 2.10.4) [START_REF] Smart | Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER[END_REF] . Manual building was performed with Coot (version 0.9.6.

SEC-SAXS data measurement

All the data was collected in SEC-SAXS mode with an in-line Superdex 200 Increase 5/150 GL column (GE Healthcare).

Homogeneous reconstituted proteins were loaded on our anaerobic setup which was based on an HPLC system equilibrated with degassed buffer containing 1 mM DTT and an auto-sampler flushed by a continuous flux of N2. The SAXS data was collected at the SWING beamline at the SOLEIL synchrotron, France, equipped with an Eiger 4M detector with a sample-to-detector distance of 2 m 72 . The parameters used for SAXS data collection are given in Supplementary Table 10. More information is available in Supplementary Table 10.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.