Structure and Function of a Class III Metal-Independent Lanthipeptide Synthetase

In bacteria, Ser/Thr protein kinase-like sequences are found as part of large multidomain polypeptides that biosynthesize lanthipeptides, a class of natural products distinguished by the presence of thioether cross-links. The kinase domain phosphorylates Ser or Thr residues in the peptide substrates. Subsequent β-elimination by a lyase domain yields electrophilic dehydroamino acids, which can undergo cyclase domain-catalyzed cyclization to yield conformationally restricted, bioactive compounds. Here, we reconstitute the biosynthetic pathway for a class III lanthipeptide from Bacillus thuringiensis NRRL B-23139, including characterization of a two-component protease for leader peptide excision. We also describe the first crystal structures of a class III lanthipeptide synthetase, consisting of the lyase, kinase, and cyclase domains, in various states including complexes with its leader peptide and nucleotide. The structure shows interactions between all three domains that result in an active conformation of the kinase domain. Biochemical analysis demonstrates that the three domains undergo movement upon binding of the leader peptide to establish interdomain allosteric interactions that stabilize this active form. These studies inform on the regulatory mechanism of substrate recognition and provide a framework for engineering of variants of biotechnological interest.


Cloning, Heterologous Expression and Purification of ThurKC Proteins
Primers used for polymerase chain reaction (PCR) amplification are listed in Table S2.The gene encoding ThurKC (WP_172554310.1) was amplified from Bacillus thuringiensis sv.
andalousiensis NRRL B23139 genomic DNA using primers designed based on the published sequence.The amplified PCR product was inserted into linearized pET His 6 tobacco etch virus (TEV) protease LIC cloning vector or pET His 6 MBP TEV LIC cloning vector (gifts from Scott Gradia, Addgene plasmid #29666 and #29656, respectively) using the Gibson Assembly Kit from New England Biosciences.Gibson assembled plasmids were maintained and propagated in Escherichia coli DH10β and the integrity of the gene was confirmed using dideoxy DNA sequencing.For protein production, the sequence-verified plasmids vectors were transformed into E. coli Rosetta (DE3) (Novagen) and plated on LB-Agar plates with ampicillin at 143 µM and chloramphenicol at 62 µM for the linearized pET His 6 TEV LIC ThurKC clone, and kanamycin at 52 µM and chloramphenicol at 62 µM for the pET His 6 MBP TEV LIC ThurKC clone.Single colonies were inoculated into LB media supplemented with the appropriate antibiotics grown at 37 °C to an OD 600 of 0.6 before cooling in an ice bath ice for 15 minutes.Protein production was induced with the addition of 500 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were allowed to grow for additional 18 hours at 18 o C. Following growth, cells were harvested by centrifugation (3500 rpm, 20 min, 4 °C) and resuspended in suspension buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 10% glycerol).Attempts to grow ThurKC alone resulted in low levels of protein expression with numerous degradation fragments.
The gene for ThurA 1 was cloned into MCS2 of pRSFDuet-1 (Novagen plasmid #71341-3) and cotransformed with pET His 6 TEV LIC ThurKC into E. coli Rosetta (DE3) with the appropriate antibiotics (ampicillin at 143 µM, kanamycin at 52 µM, and chloramphenicol at 62 µM).Protein expression and production was carried out as described above, and cells were harvested by centrifugation and resuspended in suspension buffer.Cells were lysed by sonication with 30 second cycles at 40% amplitude with 1 min resting intervals, for a total of seven cycles.The crude lysate was centrifuged for 40 minutes at 14000 rpm and 4 °C to clear insoluble cell debris.The cleared lysate was passed through a 5 ml HisTrap nickel-nitrilotriacetic acid (Ni-NTA) affinity column that was equilibrated with the suspension buffer.The column was washed with 50 mL of Wash Buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, and 30 mM imidazole).ThurKC was eluted on an ÄKTA prime system (GE Healthcare Life Sciences) with a linear gradient over 40 mL, from 25 mM to 250 mM imidazole, at a flow rate of 2 mL min -1 .The purity of the eluted fractions was determined using SDS-PAGElectrophoresis.Pure fractions containing the protein were dialyzed overnight at 4 °C into dialysis buffer (300 mM NaCl, 20 mM Tris pH 8.0 and 10% glycerol) with the addition of TEV protease to remove the His 6 tag.
Subtractive purification using the His-Trap Ni-NTA column was used to remove the His 6 affinity tag, and an SDS-PAGE was used to confirm the purity of the tag-free protein samples.Proteins samples were concentrated in a 50 kDa Amicon (Millipore Sigma) and then further purified by size exclusion chromatography on a Superdex 200 column (GE Healthcare Lifesciences) equilibrated with gel filtration buffer (300 mM KCl and 20 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) at pH 7.5).The protein was further concentrated with a 50 kDa Amicon and protein concentration was measured using a Nanodrop.
For production of the single chain fusion of the leader peptide with ThurKC, the ThurA 1 leader sequence was amplified using two rounds of PCR, one to add the SGSGSGS linker and the second to add an overhang designed for LIC cloning.The ThurA 1 Leader-SGSGSGS sequence was inserted into a linearized pET His 6 TEV LIC ThurKC vector between the affinity tag and the ThurKC coding sequence (hereafter LP-GS x -ThurKC).The integrity of the plasmid was verified by DNA sequencing and then transformed into E. coli Rosetta (DE3) for protein expression as described above, with ampicillin at 143 µM and chloramphenicol at 62 µM.Different primers were used to extend the linker between the ThurA1 leader and ThurKC, with no discernible differences in protein yields.Selenomethionine incorporated LP-ThurKC was produced in minimal media using the method of metabolic suppression and purified in the same manner as described above for the pET His 6 TEV LIC ThurKC and pRSFDuet-1 MCS2 ThurA 1 constructs. 1

Cloning, Expression and Purification of Modified ThurA 1 Peptide
The ThurA 1 insert was cloned from the genomic DNA of Bacillus thuringiensis sv.andalousiensis NRRL B23139 and ligated into a linearized pET28 MBP His 6 Thrombin-TEV vector (using a Gibson Assembly Kit.The sequence-confirmed plasmid was co-transformed along with pET MBP LIC ThurKC into E. coli BL-21 Rosetta for production of the peptide.The MBP His 6 -tagged ThurA 1 peptide was co-expressed with MBP-tagged ThurKC with ampicillin at 143 µM, kanamycin at 52 µM, and chloramphenicol at 62 µM as described and purified using a 5 ml Ni-NTA affinity column.Fractions were checked for purity using an SDS-PAGE where the MBP His 6 ThurA 1 co-eluted with the MBP-tagged ThurKC.The complex was concentrated using an 30kDa Amicon (Millipore Sigma) to a final concentration of 30 g -1 L -1 .
The concentrated complex was incubated for 20 hours at 18°C with TEV and 5 mM ATP and 5mM MgCl 2 to induce complete modification of the peptide.Upon cleavage of the MBP His 6 tag, a Ser-Gly-Ser was retained at the N-terminus of the peptide.After tag cleavage, 1 eq. of 50% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA) was added to the reaction to precipitate MBP, TEV, and MBP-ThurKC.After centrifugation to remove the insoluble proteins, the supernatant was purified by HPLC using a VP NUCLEODUR 250 x10 mm, 5 µm, C8 column and a Shimadzu LC-20AD HPLC system.Chromatographic separation utilized a linear gradient (solvent A composed of H 2 O and 0.1% Trifluoroacetic acid and solvent B composed of ACN and 0.1% TFA), starting at 30% B and going to 65% B over 40 minutes.Fractions were monitored by absorbance at 214 or 280 nm, and collected fractions were mixed in a 1:1 ratio with 2,5-dihydroxybenzoic acid matrix, spotted on a Bruker MALDI plate, and analyzed by MALDI-TOF-MS in a Bruker Daltonics UltrafleXtreme MALDI TOF/TOF instrument (Bruker).Fully modified ThurA 1 peptides, corresponding to 11 dehydrations and intermediates were identified by MALDI-TOF-MS and lyophilized for further use.Similar methods were used for the purification of modified ThurA 7 peptide.

Cloning, Expression and Purification of the ThurA 1 Leader Peptide
The ThurA 1 leader sequence was cloned from the genomic DNA of Bacillus thuringiensis sv.
andalousiensis NRRL B23139 and ligated into a linearized the pET His 6 MBP TEV vector.The sequence-confirmed plasmid was plasmid was transformed into E. coli BL21 Rosetta for heterologous production as above with kanamycin at 52 µM and chloramphenicol at 62 µM.
Following IPTG induction, cells were harvested by centrifugation (3500 rpm, 40 min, 4 °C) and resuspended in denaturing suspension buffer (8M Urea, 50 mM Tris pH 8.0, 150 mM NaCl).Cells were lysed by sonication with 30 second cycles at 40% amplitude with 1 min resting intervals, for a total of seven cycles.The crude lysate was centrifuged for 60 minutes at 14000 rpm and 4 °C to clear insoluble cell debris.The pET His 6 MBP tagged ThurA 1 leader peptide was purified using a 5 ml Ni-NTA affinity column under denaturing conditions.Purified fractions were concentrated to 30 g -1 L -1 using a 30 kDa Amicon (Millipore Sigma) and dialyzed overnight into a buffer of 20 mM Tris pH 8.0 and 150 mM NaCl.The dialyzed fraction was incubated for 20 hours at 18°C with TEV to remove the pET His 6 MBP tag.Cleavage of the tag retained a Ser-Asn-Ala linker at the N-terminus of the peptide.
After affinity tag cleavage, 1 eq. of 50% ACN and 0.1% TFA was added to the reaction to precipitate MBP and TEV.After centrifugation to pellet the insoluble proteins, the supernatant was purified by HPLC using a VisionHT C18 HighLoad 250 x 10mm, 5µm, C18 column and a Shimadzu LC-20AD HPLC system.Chromatographic separation utilized a linear gradient (solvent A composed of H 2 O and 0.1% TFA and solvent B composed of ACN and 0.1% TFA), starting at 5% B and going to 65% B over 40 minutes.Fractions were monitored by absorbance at 214 nm, and collected fractions were mixed in a 1:1 ratio with 2,5-dihydroxybenzoic acid matrix, spotted on a Bruker MALDI plate, and analyzed by MALDI-TOF-MS in a Bruker Daltonics UltrafleXtreme MALDI TOF/TOF instrument (Bruker).Purified ThurA 1 leader peptide was lyophilized for crystallographic studies.

Cloning, Expression and Purification of Unmodified and Modified C 8 ThurA 1 peptide
Attempts to purify full-length unmodified ThurA 1 precursor failed due to proteolysis in situ.
Analysis of the fragmentation pattern of the resultant peptide suggest that the precursor peptide was cleaved at Val14 of the core and was missing the C-terminal 8 residues.A peptide encompassing all but the C-terminal 8 residues (hereafter C 8 ThurA 1 ) was cloned into pET SUMO His 6 TEV vector, and sequence-verified clones were transformed into E. coli BL21 Rosetta and grown in liquid culture with ampicillin at 143 µM and chloramphenicol at 62 µM.The unmodified C 8 ThurA 1 peptide was purified under denaturing conditions, as described for the ThurA 1 leader peptide, and lyophilized.
For production of ThurKC modified C 8 ThurA 1 , the ThurKC sequence was cloned into the pAYCDuet-1 vector for co-expression.The pACYCDuet-1 ThurKC and pET Sumo His 6 TEV C 8 ThurA 1 plasmids were co-transformed into E. coli BL21 cells for protein expression and purification as described above.

Cloning, Expression and Purification of the leader proteases ThurP1/P2 and ThurP3/P4
The sequence of ThurP1/P2 and ThurP3/P4 share sequence identity to other proteases that are implicated in leader peptide removal. 2 The two sets of proteases were each cloned from genomic DNA of Bacillus thuringiensis sv.andalousiensis NRRL B23139 as a single insert in the vector a pET His 6 TEV LIC (gift from Scott Gradia, Addgene plasmid # 29656), with ThurP3 fused to a His tag.E. coli DH10β was used for transformation and plasmid production.The insertions were verified by DNA sequencing.The confirmed plasmid was transformed into E. coli BL21 Rosetta using LB plates with ampicillin at 143 µM and chloramphenicol at 62 µM.The protease complexes were expressed and purified using the same protocol and buffers as Ni-NTA purification method of pET His 6 LIC ThurKC.Protease complex purification was confirmed by SDS-PAGE gel and the fractions containing the protease complex were dialyzed overnight at 4 °C into a buffer with 300 mM NaCl, 20 mM Tris pH 8.0, and 10% glycerol.Following dialysis, ThurP1/P2 and ThurP3/4 was concentrated down to 5 mg/mL using a 30kDa Amicon concentrator (Millipore Sigma), aliquoted, and frozen for later use.

Leader removal of ThurA 1 , ThurA 7 , and ∆C8 ThurA 1 peptides and core purification
The modified ThurA 1 was resuspended in a cleavage buffer (150 mM NaCl and 20 mM Tris pH 8.0) and protease complex was added in a ratio of peptide to protease of 1: 100, and the peptide/ protease mixture was incubated for 24 hours at 18 °C with either ThurP1/P2 or ThurP3/P4.After leader removal from the was confirmed using the ThurP3/P4 only by MALDI-TOF-MS, 1 eq. of 50% ACN and 0.1% TFA was added to the reaction to precipitate the proteases.After centrifugation to pellet the insoluble protein, the supernatant was purified by HPLC using a VisionHT C18 HighLoad 250 x 10mm, 5µm, C18 column and a Shimadzu LC-20AD HPLC system.Chromatographic separation utilized a linear gradient (solvent A composed of H 2 O and 0.1% TFA and solvent B composed of ACN and 0.1% TFA), starting at 30% B and going to 65% B over 40 minutes.Fractions were monitored by absorbance at 280 nm, and collected fractions were mixed in a 1:1 ratio with 2,5-dihydroxybenzoic acid matrix, spotted on a Bruker MALDI plate, and analyzed by MALDI-TOF-MS in a Bruker Daltonics UltrafleXtreme MALDI TOF/TOF instrument (Bruker).Purified ThurKC modified ThurA 1 core peptide was identified and lyophilized for further use.
ThurP3/P4 was also active as the functional protease for the ThurKC modified ThurA 7 and was used in an identical manner to obtain the modified core.The same methods were used for HPLC purification of the ThurA 7 modified core as for ThurA 1 .Interestingly, the ThurP3/P4 peptidases were not functional for the removal of the leader of the ThurKC modified C 8 ThurA 1.The ThurP1/P2 complex was efficient in leader removal of the C 8 ThurA 1 leader.The ThurKC modified C 8 ThurA 1 14 residue truncated core was isolated by HPLC using a VisionHT C18 HighLoad 250 x 10mm, 5µm, C18 column and a Shimadzu LC-20AD HPLC system.The chromatographic separation utilized a linear gradient (solvent A composed of H2O and 0.1% TFA and solvent B composed of ACN and 0.1% TFA), starting at 5% B and going to 60% B over 40 minutes.

ThurA 1 peptide in vivo co-expressions with mutant ThurKC
Primers used for cloning are listed in Table S2.All mutants were cloned using Site-directed, Ligase-Independent Mutagenesis (SLIM), with primers designed based on the pET MBP LIC ThurKC sequencing data.Peptide and protein expression is the same as that described in the previous section.Following the incubation of the eluted mutant ThurKC and MBP His 6 ThurA 1 overnight with TEV, the free modified ThurA 1 peptides were then clarified by centrifugation and desalted using C18 ZipTips (Millipore Sigma).The samples were analyzed by MALDI-TOF-MS for crude analysis of the peptide expression.Identical methodology was done on the mutants of ThurA 1 leader peptide leader and WT ThurKC.Analytical size exclusion was run for the WT ThurKC, lyase-kinase, cyclase, and a 1:1 ratio mixture of the latter two was run on a Superdex 200 Increase 10/300 GL column in a buffer of 40 mM HEPES on 150 mM KCl at a flow rate of 0.5 mL/min in a GE AKTA 10 UPC FPLC system.Elution traces were plotted and analyzed using OriginPro.

Chemical modification of the Dha/Dhb residues
β-mercaptoethanol adduct formation was performed according to procedures previously described. 3Andalusicin cores (ThurA 1 for A, ThurA 7 for B, 0.2 mg) was dissolved in the reaction mixture consisting of 280 μl ethanol, 200 μl water, 65 μl 5 M NaOH, and 60 μl β-mercaptoethanol and incubated at 50°C for 2 h.After the incubation was completed, the reaction mixture two-fold diluted with water and desalted using C18 ZipTips (Millipore Sigma) according to manufacturer's protocol, and subjected to MALDI-TOF-MS analysis.

Dehydrated ThurA 1 and ThurA 7 fragmentation spectra
ThurA 1 and ThurA 7 fully modified peptides were mixed 1:1 ratio with 2,5-dihydroxybenzoic acid matrix, spotted on a Bruker MALDI plate, and analyzed by MALDI-TOF-MS in a Bruker Daltonics UltrafleXtreme MALDI TOF/TOF instrument (Bruker).Spectra of fragmentation were obtained in LIFT mode; the accuracy of product ions measurement was within 1 Da range.Mass spectra were processed using FlexAnalysis 3.2 software and analyzed manually.

In vitro ThurKC Activity Assay
The purified ∆C8ThurA 1 was resuspended in 150 mM NaCl and 20 mM Tris pH 8.0 to a concentration of 200 µM.The purified ThurKC from the pET His6 TEV LIC ThurKC and pRSFDuet-1 MCS2 ThurA 1 co-expression construct was added to a 20 µM concentration, along with 5 mM and ATP and 5mM MgCl 2. The reaction was incubated for 5 hours at 25 °C and desalted using C18 ZipTips (Millipore Sigma).The samples were analyzed by MALDI-TOF-MS for analysis of the Dhb formation.

In vitro LP-ThurKC Activity Assay
The ThurA 1 9mer and ThurA 1 14mer peptide core peptides were purchased from GenScript with 90% purity and were used directly.The purified LP-GS 7 -ThurKC was added to a 20 µM concentration, along with 5 mM and ATP and 5mM MgCl 2. The reaction was incubated for 5 hours and desalted using C18 ZipTips (Millipore Sigma).The samples were analyzed by MALDI-TOF-MS for analysis of the Dhb formation.

Crystallization, Data Collection, Phasing and Refinement
Initial crystallization conditions for ThurKC incubated with 1:15 ratio to ThurA 1 leader peptide and 2 mM ATP and MgCl 2 were established using sitting drop sparse matrix screening.
Preliminary conditions were optimized to a final condition consisting of 15% glycerol, 16% PEG 6000, 80 mM sodium cacodylate pH 6.5, 160 mM calcium acetate.Diffraction quality crystals were grown by the hanging drop method.Briefly, a 1 μL solution of 18 mg/mL ThurKC and leader peptide were mixed with 1 μL of the above precipitant and incubated over a solution of the same at 9 °C.Plate-like crystals grew over 7 days and were transiently soaked in the precipitant solution supplemented with 20% glycerol immediately prior to vitrification by direct immersion into liquid nitrogen.
The data set collected in which the leader peptide is not observed was collected from a crystal grown when 1 μL of ThurKC at 18 mg/mL was incubated with 1 μL of 18% (w/v) PEG 3350, 100 mM Bis tris propane/ hydrochloric acid pH 7.5, 200 mM sodium fluoride, and 3% 2-propanol.
Plate-like crystals grew over 5 days and were transiently soaked in the precipitant solution supplemented with 25% glycerol immediately prior to vitrification into liquid nitrogen.
Attempts to obtain phases of cocrystals of ThurKC in complex with the ThurA 1 leader peptide did succeed, due to the low yields of this same protein construct in M9 media and inability to obtain anomalous signal for multiple heavy atom-soaked crystals.We created the single chain fusion construct as described in an earlier section.The LP-ThurKC was incubated at 18 mg/mL with 3 mM ATP and 3 mM MgCl 2 and screened using sitting drop sparse matrix screening.Initial conditions were optimized to 0.2 M potassium citrate tribasic monohydrate and 19 % w/v PEG 3350.Diffraction quality crystals were grown by the sitting drop method.Briefly, a 1 μL solution of 23 mg/mL of LP-ThurKC, 2 mM ATP and 2 mM MgCl 2 (1 μL) was mixed with 1 μL of the latter precipitant and incubated over a solution of the same at 9 °C.Large plate crystals grew over 7 days, and were subsequently soaked for 6 hours at 9 °C in the precipitant solution supplemented with 7 mM ATP and 7mM CaCl 2. The crystals were then transiently soaked in precipitant solution supplemented with 20% glycerol immediately prior to vitrification by direct immersion in liquid nitrogen.Selenomethionine incorporated LP-ThurKC was similarly screened using a sparse matrix screen.The best diffracting crystals were obtained by incubation of the selenomethionine derived LP-ThurKC incubated with 2 mM ATP and 2 mM MgCl 2 on two different conditions: 1) 0.09 mixture of sodium nitrate, sodium phosphate dibasic, and ammonium sulfate, 0.1 M buffers Tris base and Bicine pH 8.5, and precipitant mix of 20% v/v PEG 500 MME, 10% w/v PEG 20,000; 2) 0.2 M ammonium sulfate, 0.1 M Tris pH 8.5, and 25% PEG 3350.The crystals were vitrified as above prior to data collection.Diffraction data were collected at the Advanced Photon Source Sector-21 LS-CAT at Argonne National Labs at beamlines 21-ID-D, 21-ID-F, and 21-ID-G.All diffraction data were indexed, scaled, and integrated using autoPROC. 4Crystallographic phases were determined for LP-ThurKC using the CRANK2 5 pipeline as implemented in CCP4 6 from data collected on SeMet labelled protein crystals.An initial model was produced using Parrot and Buccaneer, 7 and this was further improved through manual model building with COOT. 8Phases for the datasets of ThurKC incubated with ThurA 1 leader were determined using Phaser, 9 using the LP-ThurKC structure as a search model.The models were subject to rounds of manual building followed by refinement using REFMAC5 10

2 .
R-factor = (|Fobs|-k|Fcalc|)/ |Fobs|and R-free is the R value for a test set of reflections consisting of a random 5% of the diffraction data not used in refinement.

Figure S3 :
Figure S3: Tandem MS analysis of reconstituted ThurA 1 .MALDI-TOF MS-MS analysis of mature ThurA 1 and structure proposed based on fragmentation pattern excluding residues 17-23.

Figure S4 :
Figure S4: Tandem MS analysis of reconstituted ThurA 7. MALDI-TOF MS-MS analysis of mature ThurA 7 and structure proposed based on fragmentation pattern excluding residues 17-23.

Figure S5 :
Figure S5: Incubation of modified core (mc) ThurA 1 or ThurA 7 after removal of their leader sequences with recombinant ThurMet methyltransferase and SAM yielded products with mass changes corresponding to the addition of two and three methyl groups.

Figure S7 :
Figure S7: Heterologous expression of ThurKC modified ∆C8 (m∆C8) ThurA 1 in E. coli and leader removal of purified peptides analyzed by MALDI-TOF MS.Although the leaders of fully modified ThurA 1 and ThurA 7 peptides is removed efficiently by ThurP3/P4, the leader of the truncated m∆C8 ThurA 1 is most efficiently removed by ThurP1/P2 (see section marked "Cloning, Expression and Purification of the leader proteases ThurP1/P2 and ThurP3/P4").

Figure S8 :
Figure S8: MALDI-TOF spectra of in vivo modified ThurA 1 core by WT ThurKC.The precursor ThurA 1 core (expected MW of 2701.27Da) is degraded in E. coli when expressed with a His6-MBP N terminal tag, as revealed following tag removal with TEV.The same His6-MBP-TEV ThurA 1 core is modified and not degraded in vitro when expressed with the fusion LP-GS 7 -ThurKC.

Figure S10 :
Figure S10: Heterologous co-expression of ThurA 1 peptide with ThurKC lyase domain variants in E. coli and analysis by MALDI-TOF MS.

Figure S11 :
Figure S11: Sequence alignment of lyase domains of class III LanKC reported to produce lanthipeptide containing labionin.The alignment was performed by the Clustal Omega tool using the order of input.Above 50% conserved in sequence alignment is highlited and image is created using Jalview software.Residues predicted to be relevant to catalytic activity are marked with an red asterisk and corresponding residue number in ThurKC sequence.Residues and loops that are directly involved with binding the ThurA 1 leader and referred to in the main text are marked with an blue asterisk or box, respectively, and labeled with corresponding residue number in the ThurKC sequence.Residues corresponding to the ThurKC N-terminal helix are boxed in yellow.

Figure S12 :
Figure S12: Comparison of the kinase domains of PIM-1 Kinase and ThurKC.(A) The ThurKC kinase domain (pink, residues shown as sticks) bound to ATP (turquoise) and Ca 2+ ion (green) is compared with (B) PIM-1 kinase (purple, residues shown as sticks) bound to AMP-PNP (PDB 1YXT).The equivalent loop activation loop is highlighted, with DFE/DFG motif and APE motif residues shown as sticks.

Figure S13 :
Figure S13: Sequence alignment of kinase domains of class III LanKC reported to produce lanthipeptide containing labionin.The alignment was performed by the Clustal Omega tool using the order of input.Above 50% conserved in sequence alignment is highlited and image is created using Jalview software.Above 50% conserved in sequence alignment is highlited.Conserved residues predicted to be relevant to catalytic activity are marked with an asterisk and corresponding residue number in ThurKC sequence.Residues that are directly involved with binding the ThurA 1 leader and referred to in the main text are marked with an blue asterisk and labeled with corresponding residue number in the ThurKC sequence.The activation loop, ranging from D382 to K408 is boxed in purple, the catalytic loop V360 to N369 is boxed in green, and the loop preceding the αC helix (P261 to D271) that aids in activation loop positioning is boxed in orange.

Figure S15 :
Figure S15: Comparison of the cyclase domains of LanCL1 and ThurKC.(A) Structure of the eukaryotic cyclase homolog LanCL1 bound to glutathionine and a substrate peptide derived from Erk. (B) An Alphafold model of ThurKC in complex with the full-length ThurA 1 peptide showing a close up view of the cyclase domain.Residues implicated in catalysis and tested biochemically are shown as stick figures.

Figure S16 :
Figure S16: Alphafold active site predictions of LanKCs that have been biochemically characterized to produce Labionin rings.The classic zinc binding amino acid residues in NisC, Cys284, Cys330, and His331, are replaced by Ser726, Phe770, and Asp771 in ThurKC when the cyclase domains are superimposed.Alphafold is able to predict the stucture of ThurKC with a prediction confidence (predicted LDDT score) of 92.33, and positions the latter 3 residues in the same position as the LP-SG7-ThurKC X-ray structure bound to ATP and Ca 2+ .

Figure S17 :
Figure S17: Sequence alignment of cyclase domains of class III LanKC reported to produce lanthipeptide containing labionin.The alignment was performed by the Clustal Omega tool using the order of input.Above 50% conserved in sequence alignment is highlited and image is created using Jalview software.Conserved residues predicted to be relevant to catalytic activity are marked with an asterisk and corresponding residue number in ThurKC sequence.Loops that are predicted to orient the ThurA 1 leader and referred to in the main text are marked with an blue box and labeled with corresponding residue number in the ThurKC sequence.

Figure S18 :
Figure S18: Alphafold predictions suggest conservation of crucial residues in the cyclase domain loop region of labionin producing LanKCs.A sequence alignment for the cyclase domains of LanKC enzymes reported to produce labionin rings did not reflect conservation of residues potentially important for catalysis.Superimpositions of Alphafold predictions of these enzymes show that although the residues may not be directly superimposable, they are present in the nearby loop region in the cyclase active site cavity.The 4 loop regions are color coded.Residues directly superimposing the original ThurKC residues in other Class III Lanthionine synthetases are to their right.If the residue is conserved, it is bolded.If the residue is conserved somewhere else in that loop region, it is bold and italics.

Figure S19 :
Figure S19: Leader binding region of LP-SG7-ThurKC.ThurA 1 leader peptide ranging from Asp(-10) to Met(-22) is shown as a green ribbon and amino acid side chains are shown as sticks, and the residues in the kinase and lyase domains interacting with them are in pink.

Figure S20 :
Figure S20: Heterologous co-expression of ThurA 1 peptide leader mutants with WT ThurKC in E. coli and analysis by MALDI-TOF MS.

Figure S21 :
Figure S21: Basis for orientation of the core peptide.Structural features from each domain may help direct the core peptide towards the active site(s) and away from solvent.

Figure S22 :
Figure S22: MALDI-TOF MS analysis of ThurA 1 precursor peptide co-expressed with wildtype and truncated ThurKC.Wild-type ThurKC can effectively modify the full-length precursor peptide to produce mThurA 1 with 11 dehydrations, but co-expression of the full-length precursor with the Lyase-Kinase did not result in any modifications.

Table S4 .
and PHENIX.Refine 11 until convergence Crystallographic statistics are listed in

Table S2 :
Primers synthetized for this study.

Table S3 :
Crystallographic Statistics for structures of ThurKC obtained in this study.1. Highest resolution shell is shown in parenthesis.