Chemistry & Biology 13 Supplemental Data Engineering Dehydro Amino Acids and Thioethers into Peptides Using Lacticin 481 Synthetase Champak Chatterjee, Gregory C. Patton, Lisa Cooper, Moushumi Paul, and Wilfred A. van der Donk General PCR Methods for Preparation of LctA Mutants Site-Directed Mutagenesis via Mega Primer PCR: PCR amplifications were performed with mutant forward primers (.5 µm) and T7 terminator primer (.5 µm), deoxyribonucleotides (1 mm each), MgCl 2 (1.5 mm), and 1x PCR rxn buffer (-MgCl 2 ). The template was isolated from E. coli DH5α cells carrying the pet15b-lcta plasmid using the QIAGEN Plasmid Midi Kit. Taq DNA polymerase, Platinum Taq DNA polymerase, and/or Platinum Pfx DNA polymerase were used. Successful reactions were concentrated via centrivap from µl to less than 5 µl, then purified by gel electrophoresis (2% agarose) and isolated using QIAquick Gel Extraction Kit (QIAGEN). Linear amplification was performed with Mega Primer reverse primer. The pet15b- LctA plasmid (Xie et al., 4) was used as the template DNA unless otherwise denoted below. Taq DNA polymerase, Platinum Taq DNA polymerase, and/or Platinum Pfx DNA polymerase were used. The linear amplification included six cycles of denaturing, annealing, and extending in the absence of the forward primer. Following the linear amplification, long T7 promoter primer (5 -CGCGAAATTAATACGACTCACTATAGGGGAATTGTGAG-3,.5 µm) was added and thirty cycles of denaturing, annealing, and extending under the same conditions as the final linear amplification steps were used to amplify the desired region of DNA. Successful reactions were concentrated from µl to less than 5 µl, then purified by gel electrophoresis (2% agarose) and isolated using QIAquick Gel Extraction Kit (QIAGEN). Construction of Plasmids pet15b-lcta(1-37)s35a-intein-cbd. The partial lcta(1-37)s35a gene was amplified using the primers T7 promoter: 5 -TAATACGACTCACTATAG-3 and LctA(1-37)S35A-SapI-RP: 5 - GAATATATGCT-CTTCCGCATTCATGTGCAATTGTATGAATAAC-3 with the plasmid pet15b-lcta as the template DNA. The PCR product was gel purified, digested with NdeI and SapI restriction enzymes, and ligated into the identically digested ptxb1 vector. The resulting plasmid, ptxb1-s35a#3 was sequenced and found to contain the correct insert with a silent mutation (A33G) which did not affect the amino acid at that position (Gln11). The entire lcta(1-37)s35a-intien-cbd fusion gene was then obtained from ptxb1-s35a#3 by digestion with NdeI and BamHI, and ligated into an empty pet15b vector that had been previously digested and treated with CIP to remove the terminal 5 -phosphate groups. The resulting plasmid p15-lcta(1-37)s35a-int-cbd#1 was used to overexpress and purify the truncated His-LctA mutant, His- LctA(1-37)S35A-MESNa, as the peptide thioester.
pet15b LctA G27I/G29H. The partial lcta gene was amplified using the primers LctA G27I/G29HFP: 5 -GGTGCAAAAGGCATCAGTCATGTTATTCATACA -3 and T7 terminator: 5 -GCTAGTTATTGCTCAGCGG-3 and the plasmid pet15b-lcta was used as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 promoter primer to yield the full lcta gene. The PCR product was digested with NdeI and BamHI restriction enzymes then ligated into the pet15b vector. DNA sequencing revealed a nucleotide change (A33G) leading to a silent mutation (Q11Q) in addition to the desired mutation. pet15b LctA(K25-G26insAAA)G27I/G29H. The partial lcta gene was amplified using the primers LctA AAAGISH: 5 -TTAGGTGCAAAAGCAGCAGCAGGCATCAGTCAT -3 and T7 terminator: 5 -GCTAGTTATTGCTCAGCGG-3 and the plasmid pet15b-lcta G27I/G29H was used as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 promoter primer and pet15b LctA G27I/G29H as template DNA to yield the full lcta gene. The PCR product was digested with NdeI and BamHI restriction enzymes then ligated into the pet15b vector. DNA sequencing revealed a nucleotide change (A33G) leading to a silent mutation (Q11Q) in addition to the desired mutation. pet15b LctA(K25-G26insAAA). The partial lcta gene was amplified using the primers LctA AAAGISG: 5 -AGCAGGCGGCAGTGGAGTTATTCATACAATTC-3 and T7 terminator: 5 - GCTAGTTATTGCTCAGCGG-3 and the plasmid pet15b-lcta (K25-G26insAAA) G29H was used as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 promoter primer and pet15b-lcta (K25-G26insAAA) G29H as the template DNA to yield the full lcta gene. The PCR product was digested with NdeI and BamHI restriction enzymes then ligated into the pet15b vector. DNA sequencing revealed a nucleotide change (A33G) leading to a silent mutation (Q11Q) in addition to the desired mutation. pet15b LctA T33S. The partial lcta gene was amplified using the primers LctA T33S FP: 5 - CAGTGGAGTTATTCATTCAATTTCTCATGAATGTAAT-3 and T7 terminator: 5 - GCTAGTTATTGCTCAGCGG-3 and the plasmid pet15b-lcta was used as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 pet15b LctA S35T. The partial lcta gene was amplified using the primers LctA S35T FP: 5 - GGAGTTATTCATACAATTACTCATGAATGTATTATG-3 and T7 terminator: 5 - GCTAGTTATTGCTCAGCGG-3 and the plasmid pet15b-lcta was used as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 pet15b LctA S42T. The partial lcta gene was amplified using the primers LctA S42T FP: 5 - CATGAATGTAATATGAATACCTGGCAATTTGTATTT-3 and T7 terminator: 5-2
GCTAGTTATTGCTCAGCGG-3 and the plasmid pet15b-lcta was used as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 pet15b LctA T48S. The lcta gene was amplified using the primers LctA T48S FP: 5 - GGGAATTCCATATGAAAGAACAAAACTCTTTTAA-3 and LctA T48S RP: 5 - CGCGGATCCTTAAGAGCAGCAAGAA-3 and the plasmid pet15b-lcta was used as the template. The PCR product was digested with NdeI and BamHI restriction enzymes then ligated into the pet15b vector. DNA sequencing revealed a nucleotide change (A33G) leading to a silent mutation (Q11Q) in addition to the desired mutation. pet15b LctA T48A. The lcta gene was amplified using the primers LctA T48S FP: 5 - GGGAATTCCATATGAAAGAACAAAACTCTTTTAA-3 and LctA T48S RP: 5 - CGCGGATCCTTAAGAGCAGCATGCA-3 and the plasmid pet15b-lcta was used as the template. The PCR product was digested with NdeI and BamHI restriction enzymes then ligated into the pet15b vector. DNA sequencing revealed a nucleotide change (A33G) leading to a silent mutation (Q11Q) in addition to the desired mutation. pet15b LctA C49A. The lcta gene was amplified using the primers LctA C49A FP: 5 - GGGAATTCCATATGAAAGAACAAAACTCTTTTAA-3 and LctA C49A RP: 5 - CGCGGATCCTTAAGAGCATGCAGTA-3 and the plasmid pet15b-lcta was used as the template. The PCR product was digested with NdeI and BamHI restriction enzymes then ligated into the pet15b vector. DNA sequencing revealed a nucleotide change (A33G) leading to a silent mutation (Q11Q) in addition to the desired mutation. pet15b LctA C49S. The lcta gene was amplified using the primers LctA C49S FP: 5 - GGGAATTCCATATGAAAGAACAAAACTCTTTTAA-3 and LctA C49A RP: 5 - CGCGGATCCTTAAGAGCAGCTAGTA-3 and the plasmid pet15b-lcta was used as the template. The PCR product was digested with NdeI and BamHI restriction enzymes then ligated into the pet15b vector. DNA sequencing revealed a nucleotide change (A33G) leading to a silent mutation (Q11Q) in addition to the desired mutation. pet15b LctA C49AC5A. The lcta gene was amplified using the primers LctA C49S FP: 5 - GGGAATTCCATATGAAAGAACAAAACTCTTTTAA-3 and LctA C49A RP: 5 - CGCGGATCCTTAAGATGCAGCAGTA-3 and the plasmid pet15b-lcta was used as the template. The PCR product was digested with NdeI and BamHI restriction enzymes then ligated into the pet15b vector. DNA sequencing revealed a nucleotide change (A33G) leading to a silent mutation (Q11Q) in addition to the desired mutation. pet15b LctA T33A/S35A. The partial lcta gene was amplified using the primers LctAT33A/S35A FP: 5 -GGAGTTATTCATGCAATTGCTCATGAATGTAAT -3 and T7 terminator: 5 -GCTAGTTATTGCTCAGCGG-3 and the plasmid pet15b-lcta was used as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 promoter primer to yield the full lcta gene. The PCR product was digested with NdeI 3
and BamHI restriction enzymes then ligated into the pet15b vector. DNA sequencing revealed a nucleotide change (A33G) leading to a silent mutation (Q11Q) in addition to the desired mutation. pet15b-lcta H32D. The partial lcta gene (79-176) was amplified using primers LctAH32D FP: 5 -GGCAGTGGAGTTATTGATACAATTTCTCATGAATGT-3 and T7 terminator: 5 - GCTAGTTATTGCTCAGCGG-3 with the plasmid pet15b-lcta as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 pet15b-lcta I34D/H36R. The partial lcta gene (85-176) was amplified using primers LctAI34D/H36R FP: 5 -GGAGTTATTCATACAGATTCTCGTGAATGTAATATG-3 and T7 terminator: 5 -GCTAGTTATTGCTCAGCGG-3 with the plasmid pet15b-lcta as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 pet15b-lcta H36R. The partial lcta gene (85-176) was amplified using primers LctAH36R FP: 5 -GGAGTTATTCATACAATTTCTCGTGAATGTAATATG-3 and T7 terminator: 5 - GCTAGTTATTGCTCAGCGG-3 with the plasmid pet15b-lcta as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 pet15b-lcta N41D. The partial lcta gene (16-176) was amplified using primers LctAN41D FP: 5 -CATGAATGTAATATGGATAGCTGGCAATTTGTA-3 and T7 terminator: 5 - GCTAGTTATTGCTCAGCGG-3 with the plasmid pet15b-lcta as the template. The generated Mega Primer was subsequently used as the terminator primer with the long T7 Overexpression and Purification of His 6 -LctA Mutants All LctA mutants were expressed as insoluble N-terminally His 6 -tagged peptides. E. coli BL21 (DE3) cells carrying pet15b-lcta mutant plasmids were grown in LB medium containing µg/ml ampicillin at 37 o C. Cultures were induced with 1 mm IPTG at OD nm =.5.7, and grown for an additional three hours. Cells were harvested by centrifugation at 4 o C for 3 min at 11,9 x g (8 krpm) in a Beckman JLA 1.5 rotor and the cell paste was stored at - o C until use. To remove superfluous proteins, the cell paste was resuspended in LctA Start Buffer ( mm NaH 2 PO 4, ph 7.5,.5 mm imidazole, 5 mm NaCl, % glycerol) and lysed by sonication (35% Amp, 4. sec pulse, 9.9 sec pause, 1 min). The sample was centrifuged at 4 o C for 3 min 4
at 23,7 x g (14 krpm) in a Beckman JA- rotor and the supernatant was discarded. This wash was repeated. The supernatant from the second wash was discarded and the resulting cell pellet was resuspended in denaturing LctA Buffer 1 (6 M guanidine hydrochloride, mm NaH 2 PO 4, ph 7.5,.5 mm imidazole, 5 mm NaCl). The insoluble protein was removed by centrifugation and the supernatant was filtered through a.45 µm syringe filter directly onto a Ni-NTA (1 ml resin) column charged with.1 M NiSO 4 and equilibrated with LctA Buffer 1. After the supernatant was loaded, the resin was washed with LctA Buffer 2 (4 M guanidine hydrochloride, mm NaH 2 PO 4, ph 7.5, 3 mm imidazole, 3 mm NaCl). Two methods were used to elute the protein: (1) with LctA EDTA Elution Buffer (4 M guanidine hydrochloride, mm Tris, ph 7.5, 5 mm EDTA, mm NaCl) or (2) with LctA Imidazole Elution Buffer (4 M guanidine hydrochloride, mm Tris, ph 7.5, mm imidazole, mm NaCl). The elution fractions were concentrated via centrivap to less than 15 ml if necessary. Samples were filtered through.45 µm syringe filtered, and then further purified by RP-HPLC (Waters, C4 Column). Fractions were lyophilized and stored at - o C. Peptides were analyzed for purity by MALDI-TOF mass spectrometry. Expressed Protein Ligation with Cysteine Analogues The commercially available oxidized cysteine analogues, L- and D-homocystine (Sigma, St. Louis), as well as the synthetic (R)-β 3 -, and (S)-β 3 -homocystines were dissolved in ligation buffer consisting of mm HEPES, ph 7.75, mm NaCl, 1 mm EDTA, 5 mm TCEP, and 5 mm MESNa, to a final concentration of 22-37 mm. The resultant solutions were incubated at 25 C for 3 min to generate the free thiol form of the analogues. In the case of D-cysteine (Sigma, St. Louis), reduction prior to ligation was not necessary, and the ligation buffer consisted of 114 mm D-cysteine, mm HEPES, ph 7.75, mm NaCl, 1 mm EDTA and 5 mm MESNa. The cysteine analogue solutions were directly added to the lyophilized peptide thioester (Chatterjee et al, 5) to obtain a final concentration of ~1 mm of the thioester. The ph was adjusted to 7.6-7.8, and reaction was undertaken at 4 C for 16-72 h. The crude products were analyzed by MALDI-TOF MS for completeness of the reaction prior to acidification with 5% TFA. The acidified ligation mixture was incubated with mm TCEP for 3 min at 25 C prior to purification by C4 analytical RP-HPLC. Fractions containing the ligation products were analyzed by MALDI-TOF MS. LctA(1-38)S35A/C38β 3 -R-Cys. MALDI-TOF MS calculated 4126 (M). Observed, 4126 (M). LctA(1-38)S35A/C38D-Hcys. MALDI-TOF MS calculated 4126 (M). Observed, 4127 (M+1). LctA(1-38)S35A/C38β 3 -S-Cys. MALDI-TOF MS calculated 4126 (M). Observed, 4126 (M). LctA(1-38)S35A/C38D-Cys. MALDI-TOF MS calculated 4112 (M). Observed, 4113 (M+1). His 6 -LctA(1-38)S35A/C38L-Hcys. MALDI-TOF MS calculated 6159 (M). Observed, 61 (M+1). Assays with LctM and MS Analysis The HPLC purified His 6 -LctA mutant peptides were dissolved in Millipore water to give a concentration greater than 2 mg/ml. The peptides were incubated with 5 mm Tris-HCl, ph 7.5, 1 mm MgCl 2, 25 µg/ml BSA, 2.5 mm ATP, and His 6 -LctM at 25 o C for at least 4 h (total assay 5
volume was µl). Control assays were also prepared under the same conditions listed above except no His 6 -LctM was added. In some cases it was necessary to incubate peptides with 5 mm TCEP (Fluka) to reduce disulfide bonds prior to His 6 -LctM addition. After 2 h, the assays were quenched with 5 µl of 5 % TFA. The assays were then analyzed by MALDI-TOF MS at the Mass Spectrometry Laboratory from the School of Chemical Sciences at UIUC by either mixing 1 µl of sample with 9 µl of sinapic acid pre-dissolved in MeCN:H 2 O (1:1.5) in.5 % TFA or zip tipping the samples utilizing Millipore 1 µl C18 tips and eluting in 4 µl of α-hydroxyl cinnamic acid pre-dissolved in MeCN:H 2 O (1:1) in.5 % TFA. A sample of 2 µl was applied to the MALDI target and analyzed. The outcome of the assays is provided below: His 6 -LctA T48S. MALDI-TOF MS calcd. 7696 (M), 7624 (M-72), found 7691 (M), 7625 (M- 72). His 6 -LctA T48A. MALDI-TOF MS calcd. 76 (M), 7626 (M-54), found 7679 (M), 7625 (M- 54). His 6 -LctA C49A. MALDI-TOF MS calcd. 7678 (M), 78 (M-72), found 7678 (M), 75 (M- 72). His 6 -LctA C49S. MALDI-TOF MS calcd. 7694 (M), 7622 (M-72), 74 (M-9), found 7694 (M), 7618 (M-72), 71 (M-9). His 6 -LctA C49A/C5A. MALDI-TOF MS calcd. 7645 (M), 7573 (M-72), found 7573 (M-72), 7653 (M-72+). His 6 -LctA H32D. MALDI-TOF MS calcd. 7687 (M), 7615 (M-72), found 7659 (M-32), 76 (M-72), 77 (M-72+79). His 6 -LctA I34D/H36R. MALDI-TOF MS calcd. 7731 (M), 7659 (M-72), found 7675 (M-54), 7658 (M-72), 7738 (M-72+79). His 6 -LctA H36R. MALDI-TOF MS calcd. 7729 (M), 7657 (M-72), found 7676 (M-54), 7658 (M-72), 7739 (M-72+79). His 6 -LctA N41D. MALDI-TOF MS calcd. 7711 (M), 7639 (M-72), found 7671 (M-32), 7655 (M-54), 7637 (M-72), 7718 (M-72+79). His 6 -LctA T33S. MALDI-TOF MS calcd. 7696 (M), 7624 (M-72), found 7677 (M-18), 7662 (M- 32), 7644 (M-54), 7629 (M-72). His 6 -LctA S35T. MALDI-TOF MS calcd. 7724 (M), 7652 (M-72), found 77 (M), 773 (M-18), 7686 (M-32), 7668 (M-54), 7652 (M-72). His 6 -LctA S42T. MALDI-TOF MS calcd. 7724 (M), 7652 (M-72), found 7729 (M), 778 (M-18), 7693 (M-32), 7675 (M-54), 7657 (M-72). His-LctM assay with His-LctA T33A/S35A. MALDI-TOF MS calculated. 7663 (M), 7627 (M- 32), found 7663 (M), 7646 (M-18), 7629 (M-32). His 6 -LctA G27I/G29H. MALDI-TOF MS calcd. 7846 (M), 7774 (M-72), 7756 (M-9), found 7793 (M-54), 7777 (M-72). His 6 -LctA(K25-G26insAAA)G27I/G29H. MALDI-TOF MS calcd. 59 (M), 7987 (M-72), 7969 (M-9), found 7986 (M-72), 7968 (M-9). His 6 -LctA(K25-G26insAAA). MALDI-TOF MS calcd. 7923 (M), 7851 (M-72), 7833 (M-9), found 7847(M-72). 6
LysC Cleavage and MS Analysis For His 6 -LctA, three fragments are expected after treatment with LctM and LysC: 1) GSSHHHHHHSSGLVPRGSHMK, [His 6-2] MALDI-TOF MS calcd. 231 2) EQNSFNLLQEVTESELDLILGAK, [3-25] MALDI-TOF MS calcd. 2591 3) GGSGVIHTISHECNMNSWQFVFTCCS, [26-51] MALDI-TOF MS calcd. 2794 LysC Cleavage Products from His-LctM Assay with His-LctA H32D. MALDI-TOF MS calcd. 231 [His 6-2], 2591 [3-25], 2751 [26-51], found 2594 [3-25], 2754 [26-51]. LysC Cleavage Products from His-LctM Assay with His-LctA H36R. MALDI-TOF MS calcd. 231 [His 6-2], 2591 [3-25], 2792 [26-51], found 2592 [3-25], 2794 [26-51]. LysC Cleavage Products from His-LctM Assay with His-LctA T33S. MALDI-TOF MS calcd. 231 [His 6-2], 2591 [3-25], 2759 [26-51], found 2596 [3-25], 2764 [26-51] LysC Cleavage Products from His-LctM Assay with His-LctA S35T. MALDI-TOF MS calcd. 231 [His 6-2], 2591 [3-25], 2788 [26-51], found 2596 [3-25], 2792 [26-51] LysC Cleavage Products from His-LctM Assay with His-LctA S42T. MALDI-TOF MS calcd. 231 [His 6-2], 2591 [3-25], 2788 [26-51], found 2598 [3-25], 2794 [26-51] LysC Cleavage Products from His-LctM Assay with His-LctA T48A. MALDI-TOF MS calcd. 231 [His 6-2], 2591 [3-25], 2761 [26-51], found 2596 ([3-25], 2766 [26-51] Cyanylation Protocol. The LctM assay products were purified by C4 analytical RP-HPLC. Fractions containing the assay products were evaporated to dryness and to each 1.7 ml Eppendorf tube containing assay product, or an equivalent amount of the unmodified substrate, was added 5 µl of denaturation buffer ( mm sodium citrate, ph 3., 6 M guanidine hydrochloride) and 1 µl of reducing buffer ( mm sodium citrate, ph 3., mm TCEP). The substrate and assay product were redissolved by vortexing briefly and incubated at 25 C for 15 min to reduce disulfide bonds. Then µl of a freshly prepared cyanylation solution ( mm sodium citrate, ph 3., mm CDAP) was added to each tube. After an additional 15 min incubation at 25 C, the samples were acidified with 3 µl of 5% aqueous TFA. The acidified samples were purified by means of C18 Zip-tip and eluted with 3 µl of α-hydroxycinnamic acid matrix (prepared in 5% MeCN, containing.1% TFA) of which 1 µl was spotted directly on the MALDI target and analyzed by MALDI-TOF. Confirmation of Enzyme-Catalyzed Cyclization Using a LctM Mutant Although much effort was made to prevent complications of non-enzymatic cyclization by truncating the substrate and mutating Ser 35 to Ala, thus removing any Dha residues, an unambiguous control experiment was sought in which a dehydrated substrate could be incubated under the same reaction conditions in the absence of enzyme. For this purpose, an LctM mutant was needed that would retain dehydration activity but lacked any cyclization activity. The C- terminus of LctM has sequence homology with NisC, the cyclase involved in nisin biosynthesis. NisC is a Zn protein (Okeley et al. 3) and its structure was recently solved revealing the Zn ligands that are also conserved in LctM (Li et al. 6). The zinc is believed to activate the thiol of the substrate for nucleophilic attack onto the dehydro amino acid. Therefore, the conserved Zn ligand Cys836 in LctM was mutated to Ala. As intended, this mutant retained dehydration activity but did not have full cyclization activity since a free thiol was detected in the dehydrated 7
truncated product using the CDAP cyanylation protocol (M. Paul, G.C. Patton & W.A. van der Donk, manuscript in preparation). With this cyclization deficient LctM mutant in hand nonenzymatic cyclization could be ruled out for the cyclization experiments with wt LctM. 8
Figure S1. MALDI-TOF Mass Spectra of (A) LctA-S42T (7724 Da, Blue) and after Treatment with LctM (7652 Da, Red), and (B) LctA-G27I/G29H (7846 Da, Blue) and after Treatment with LctM (7774 Da, Red). (A) 7 75 7 77 7 79 (B) M-4H 2 O M Relative Intensity 7 7 8 8 9
Figure S2. MALDI-TOF Mass Spectra of LctA-K25-G26insAAA (7923 Da, Blue) and after Treatment with LctM (7851 Da, Red) M - 4H 2 O M Relative Intensity 7 7 8 8 1
Figure S3. MALDI-TOF Mass Spectra of (A) LctA(1-38)S35A/C38β 3 -R-Cys (4126 Da, blue) and after treatment with LctM (418 Da, red). (B) LctA(1-38)S35A/C38D-Hcys (4127 Da, blue) and after treatment with LctM (419 Da, red). (C) LctA(1-38)S35A/C38β 3 -S-Cys (4126 Da, blue) and after treatment with LctM (418 Da, red). (D) LctA(1-38)S35A/C38D-Cys (4113 Da, blue) and LctM assay product (95 Da, red). Met(O) oxidation products produced during the MS experiment are denoted by asterisks. (B) 395 5 4 415 425 43 395 5 4 415 425 43 395 5 4 415 425 43 (C) (D) 5 4 415 39 395 5 4 415 425 43 11
Figure S4. Cyanylation Assays on Substrates with Nonproteinogenic Amino Acids (A) His 6 -LctA(1-38)S35A/C38L-Hcys substrate (61 Da, green), after treatment with CDAP (6185 Da, blue), and after treatment with LctM and subsequently CDAP (6142 Da, red). Cyanylated product that would be formed if a free thiol were present in the assay product is indicated by a black bar (6167 Da). Unreacted substrate in the assay product was seen to be completely cyanylated (6185 Da, red) (B) LctA(1-38)S35A/C38β 3 -S-Cys substrate (4152 Da, blue) and after treatment with LctM and subsequently CDAP (419 Da, red). (C) LctA(1-38)S35A/C38D-Cys substrate (4138 Da, blue) and after treatment with LctM and subsequently CDAP (95 Da, red). In (B) and (C) cyanylated products that would be formed if a free thiol were present in the assay product is indicated by a black bar. (A) (C) 61 61 61 61 6 39 395 5 4 415 425 43 (B) 5 4 415 12
Supplemental References Chatterjee, C. et al. Lacticin 481 Synthetase Phosphorylates its Substrate during Lantibiotic Production. J. Am. Chem. Soc. 127, 15332-15333 (5). Li, B. et al. Structure and Mechanism of the Lantibiotic Cyclase Involved in Nisin Biosynthesis. Science 311, 1464 (6). Okeley, N.M., Paul, M., Stasser, J.P., Blackburn, N. & van der Donk, W.A. SpaC and NisC, the Cyclases Involved in Subtilin and Nisin Biosynthesis, are Zinc Proteins. Biochemistry 42, 13613-13624 (3). Xie, L. et al. Lacticin 481: in vitro reconstitution of lantibiotic synthetase activity. Science 33, 679-681 (4). 13