Supplementary Results
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1 upplementary Results upplementary Table 1. Primers used in this study. upplementary Figure 1. PCP3-TE assay in the absence of ul. upplementary Figure 2. Multiple alignment of the sulfazecin TE, highlighting catalytic triad components. upplementary Figure 3. Time course of tripeptide holo-pcp3 sulfonation by ul. upplementary Figure 4. Module 3 assay in the absence of ul. upplementary Figure 5. In vitro reconstitution of module 3 β-lactam formation activity showing production of desmethoxylsulfazecin by the TE domain of ulm and subsequent hydrolysis. upplementary Figure 6. Time course of module 3 production of desmethoxylsulfazecin and subsequent hydrolysis of desmethoxylsulfazecin at extended time points. upplementary Figure 7. In vitro production of sulfazecin from D-Glu D-Ala holo-pcp2. upplementary Figure 8. ul and ulp assay producing sulfazecin from desmethoxylsulfazecin. upplementary Figure 9. ydrolysis of desmethoxylsulfazecin during in vitro production of sulfazecin from D-Glu D-Ala holo-pcp2 and module 3 in vitro β-lactam production. upplementary Figure 1. ydrolysis of sulfazecin by PCP3-TE. upplementary Figure 11. ydrolysis of desmethoxylsulfazecin by PCP3-TE. upplementary Figure 12. D-PAGE gels of all proteins used in this study. upplementary Figure 13. FPLC traces of all proteins used in this study. upplementary Figure 14. Mass spectra of all proteins used in this study. upplementary ote. Preparation and characterization of synthetic compounds used in this study. ature Chemical Biology: doi:1.138/nchembio.2526
2 upplementary Table 1 Primers used in this study. Protein equence (5 3 ) PCP2 Fwd: GGCATATGGTGGCACCGCGCGACGATACCGAG Rev: GGAAGCTTTCACTGGCGACGCACGTTCGACGCGAG PCP3 Fwd: GGCATATGACGCCGGTCGAGGAACGTCTGACG Rev: GGAAGCTTTCACTCTTCCGATGCGTGTTCCAGATG M3 Fwd: GGGCATATGGAGCAGCGGCAGGTGAGCCGCGGC Rev: GGGAAGCTTTGCCGTCACACCTT M3 TE* Fwd: ATTCGCGCAGGCGAACGGCTCGGCG C2818A Rev: ACGATGGCCGGTAGGTCG M3 TE* Fwd: ATTCGCGCAGAGCAACGGCTCGG C2818 Rev: ACGATGGCCGGTAGGTCG ul Fwd: GGCATATGACAACCAACGACCATCGCATCGCA Rev: GGAAGCTTGCTCCCGATAGTTTGAATGGCTGG ul Fwd: GGCATATGGAAACACTGGACATCCGCAGGTTG Rev: GGAAGCTTGATTGAGGAAGCGGCCGTGGCCAC ulp Fwd: GGCATATGTCAAATCAGTTAATCCAGTCGAAC Rev: GGAAGCTTCGCACGGCTGCCGCGTGCCACCCA PCP3-TE Fwd: GGCATATGGAACGTCTGACGGAGATATTCCGCGGCG Rev: GGGAAGCTTTGCCGTCACACCTT PCP3-TE* Fwd: ATTCGCGCAGGCGAACGGCTCGGCG C2818A Rev: ACGATGGCCGGTAGGTCG PCP3-TE* Fwd: ATTCGCGCAGAGCAACGGCTCGG C2818 Rev: ACGATGGCCGGTAGGTCG ature Chemical Biology: doi:1.138/nchembio.2526
3 upplementary Figures Tripeptide hydrolysis tandard TE C TE C2818A TE w-t TE * Retention Time (min) Tripeptide hydrolysis product m/z = upplementary Figure 1 PCP3-TE assay in the absence of ul. hown is the extracted ion chromatogram for the tripeptide hydrolysis product shown (EIC ±.1) in each of the mutant PCP3-TE* reactions. Mutation of the TE active site cysteine to serine effects rapid generation of the hydrolysis product (calc , observed ), underscoring the importance of the cysteine active site in monobactam formation. Experimental details are listed in the nline Methods section. ature Chemical Biology: doi:1.138/nchembio.2526
4 ulte 82VFAQCGALALAIT RELVRRAD-VRALCIGGALMRTVTGKRDTR126- (92aa)-218EYAGIGYLLRDCP231 E264 73VFAQCGALAIAVA RELARRAD-VRALCVGGALMRTAIGKRDAR117- (92aa)-29EYAGVGYLLRDCP222 DM48 73VFAQCGALAIAIA RELRRKAD-LRALCIGGALMRMRGKPDTR117- (92aa)-29EYAGIGYLLRDCP222 3IL 66LGGWGGAFAYVVA EALVQGEEVLIIIDAPIPQAMEQLPRAF111-(14aa)-215RADGAFTLMQKE228 3LCR 143LAGGGVVAYEVA RELEARGLAPRGVVLIDYFDGDGGRPEEL188-(1aa)-288DWLREAAYTEG31 1MA 138VAGAGALMAYALA TELLDRGPPRGVVLIDVYPPGQDAMAWL183- (93aa)-276AWLGGG QMW 11LFGMGALLAYEVA CVLRRRGAPRPRLFVGRAPLYGDRAD145- (88aa)-233RLPGFFLGGP246 3QIT 99LVGMGAMLATAIAVRPKKIKELILVELPLPAEEKKEAVQLTTCLDYLTPQ141-(122aa)-263VFLGGLIDAA276 upplementary Figure 2 Multiple (ClustalW) alignment of the sulfazecin TE, catalytic triad components are highlighted. ulte, (P. acidophila ATCC 31363; GenBank KX75776) was aligned with TE domains from B. thailandensis (PDB: E264, GenBank: CP86.1), B. gladioli (GenBank: JPGG118.1 DM48), Aspergillus parasiticus (GenPept 3IL_A, aflatoxin biosynthesis), treptomyces sp. CK4412 (GenPept 3LCR_B, tautomycetin biosynthesis), treptomyces venezuelae (GenPept 1MA_B), treptomyces coelicolor (PDB: 3QMW, GenBank: CAA16185, prodiginine biosynthesis) Moorea producens 19L (GenPept 3QIT_A, curacin biosynthesis). ature Chemical Biology: doi:1.138/nchembio.2526
5 .8 Product : substrate Ratio Time (min) 2 3 ul PAP PAP 2 2 m/z = 1278 m/z = 1286 upplementary Figure 3 Time course of tripeptide holo-pcp3 sulfonation by ul as determined by UPLC-RM. Tripeptide holo-pcp3 (45 µm) was incubated with ul (5 µm) and PAP (3 mm) in assay buffer and monitored for 1, 5, 1, 2, 3, 6, 9, 12, 18 and 3 min. The mass of PCP3 was monitored by UPLC-RM method C. Displayed is the ion count ratio of sulfonated tripeptide holo-pcp3 (product) to unsulfonated tripeptide holo-pcp3 (substrate) which also takes into account the -glucuronidated mass for each species (-glucuronidated masses = and 1338 for the unsulfonated and sulfonated tripeptide holo-pcp3, respectively). Experiments were conducted in triplicate; data represent the mean values ± D. The error at time points of 3 min (±.9) and 6 min (±.4) are too small to be displayed. ature Chemical Biology: doi:1.138/nchembio.2526
6 Tripeptide hydrolysis standard M3 C M3 C2818A M3 w-t Retention Time (min) PCP 2 E 2 C 3 A 3 TE * Tripeptide hydrolysis product m/z = upplementary Figure 4 Module 3 assay in the absence of ul as monitored by UPLC- RM. D-Glu D-Ala as the dipeptide holo-pcp2 was condensed with diaminopropionate by module 3. The assay was carried out as described in the nline Methods, except without the addition of ul or PAP. Assay conditions are as follows: dipeptide holo-pcp2 (2 µm), holo- M3 (2 µm), ATP (2.5 mm), and L-Dap (2mM) were incubated in assay buffer for 2 hr. hown are the extracted ion chromatograms for the tripeptide hydrolysis product ( ±.1) when the assay was performed with wildtype, C2818A, and C2818 mutant module 3 constructs. Tripeptide hydrolysis product was produced in this assay when the C2818 mutant module 3 was used (calc , found ). From the results of upplementary Figure 1, it was known that mutating the TE active site to serine restores hydrolysis activity. This result indicates that condensation between the D-Glu D-Ala dipeptide and diaminopropionate occurs in the absence of sulfonation, indicating that sulfonation of the PCP3-tethered tripeptide must occur as opposed to the sulfonation of diaminopropionate-tethered PCP3. ature Chemical Biology: doi:1.138/nchembio.2526
7 a ulm Module 3 E 2 PCP 2 + C 3 A 3 TE 2 PAP ul PAP 2 Desmethoxylsulfazecin 2 3 TE ydrolysis b tandard of desmethoxylsulfazecin ydrolysis tandard M3* C2818 M3* C2818 M3* C2818A M3* C2818A M3 w-t M3 w-t Time (min) Time (min) upplementary Figure 5 (a) In vitro reconstitution of module 3 β-lactam formation activity showing production of desmethoxylsulfazecin by the TE domain of ulm and subsequent hydrolysis. (b) Extracted ion chromatograms for (left) desmethoxylsulfazecin (m/z ±.1) and (right) hydrolyzed desmethoxylsulfazecin ( ±.1) for the reaction with wildtype module 3 and module 3 TE mutants C2818A and C2818. Wildtype module 3 readily produces desmethoxylsulfazecin (calc , found ), confirmed by comparison with a synthetic standard (left). Mutation of the native cysteine active site in the ulm TE to serine (right) diverts the product profile to only simple hydrolysis (calc , found ) as confirmed by comparison with a synthetic standard. Experimental details can be found in the nline Methods. ature Chemical Biology: doi:1.138/nchembio.2526
8 β-lactam ydrolysis product Time (min) upplementary Figure 6 Module 3 production of desmethoxylsulfazecin (blue) and subsequent hydrolysis (red) of desmethoxylsulfazecin at extended time points. holo-m3 and dipeptide holo-pcp2 were generated as outlined in the nline Methods. holo-m3 (2 µm) was incubated with dipeptide holo-pcp2 (2 µm) in assay buffer supplemented with L-Dap (2 mm), ATP (2.5 mm), ul (1 µm), and PAP (1 mm). Aliquots of the reaction mixture were analyzed at 5, 45, 42, and 135 min by UPLC-RM method A. Plotted are the ion intensities at each time point for desmethoxylsulfazecin (blue) (m/z = ) and hydrolyzed desmethoxylsulfazecin (red) (m/z = ). Experiments were carried out in triplicate; data represent the mean value ± D. ature Chemical Biology: doi:1.138/nchembio.2526
9 a E 2 ulm Module 3 ul PCP 2 C 3 A 3 TE 2 PAP PAP 2 Desmethoxylsulfazecin ul Fe(II) α-kg C ulfazecin 3 ulp AM b ulfazecin tandard M3 w-t M3 C2818A Retention Time upplementary Figure 7 (a) In vitro production of sulfazecin from D-Glu D-Ala holo-pcp2. (a) Reconstitution scheme showing in vitro components necessary to produce sulfazecin. (b) UPLC- RM extracted ion chromatograms (m/z ±.1) showing (top) sulfazecin standard isolated from P. acidophila and (middle) sulfazecin production in the presence of the wildtype thioesterase (calc , found ). ulfazecin production was abolished when the thioesterase active site cysteine was mutated to alanine (Bottom). Experimental details can be found in the nline Methods. ature Chemical Biology: doi:1.138/nchembio.2526
10 ulfazecin tandard ulp ulp ulp m/z Retention Time (min) 2 Desmethoxylsulfazecin 3 ul Fe(II) α-kg 2 m/z = ul Fe(II) α-kg ulp AM 2 C 3 ulfazecin m/z = upplementary Figure 8 ul and ulp assay producing sulfazecin as determined by UPLC- RM. A synthetic standard of desmethoxylsulfazecin was incubated with either ul or both ul and ulp as outlined in the nline Methods section. hown in black is the extracted ion chromatogram for sulfazecin ( ±.1) in each reaction, plus a positive control reaction containing sulfazecin isolated from P. acidophila in assay buffer. The reaction containing both ul and ulp readily produced sulfazecin (calc , found ). The reaction mixture containing only ul produced hydroxylated desmethoxylsulfazecin (calc , found ), shown in red (extracted ion chromatogram shown in red ±.2). ature Chemical Biology: doi:1.138/nchembio.2526
11 ydrolysis tandard Tailoring Enzymes Tailoring Enzymes Retention Time (min) upplementary Figure 9 ydrolysis of desmethoxylsulfazecin during in vitro production of sulfazecin from D-Glu D-Ala holo-pcp2 and module 3 in vitro β-lactam production. Assay conditions for the two experiments are provided in the nline Methods, and were incubated for 2 hr. hown is the extracted ion chromatogram for the product of desmethoxylsulfazecin hydrolysis ( ±.1) in the M3 experiments with, and without the tailoring proteins ul and ulp. o hydrolysis of desmethoxylsulfazecin was detected in the reaction containing the tailoring proteins. owever, in the absence of the tailoring proteins, a significant amount of hydrolysis is observed (calc , found ). o hydrolysis of sulfazecin was observed. These results indicated that addition of the methoxyl group of sulfazecin protects it from hydrolysis. ature Chemical Biology: doi:1.138/nchembio.2526
12 TE w-t -TE C2818A Time (min) upplementary Figure 1 ydrolysis of sulfazecin by PCP3-TE as monitored by UPLC-RM method A. Equivalent amounts of sulfazecin slowly hydrolyzed in the presence of wildtype PCP3-TE (red) at a rate essentially identical to that in the presence of mutated TE (blue) (C2818A), which was attributed to spontaneous hydrolysis in buffer. Plotted are the ion intensities for sulfazecin (m/z = ) at the time indicated. Experimental details are provided in the nline Methods TE w-t -TE C2818A Time (min) upplementary Figure 11 ydrolysis of desmethoxylsulfazecin by PCP3-TE as monitored by UPLC-RM method A. Equivalent amounts of desmethoxylsulfazecin were rapidly hydrolyzed in the presence of wildtype PCP3-TE (red) or unaffected in the presence of a mutated TE (blue) (C2818A). Plotted are the ion intensities for desmethoxylsulfazecin (m/z = ) at the time indicated. Experimental details are provided in the nline Methods. ature Chemical Biology: doi:1.138/nchembio.2526
13 FPLC Purification % PAM 8% PAM Wildtype module Wildtype module 3 8% PAM Mutant module 3 constructs C2818A and C PCP % PAM ul % PAM 12% PAM ul % PAM 12% PAM Wildtype -TE -TE* C2818A % PAM -TE* C % PAM ulp upplementary Figure 12 D-PAGE gels of all proteins used12%inpamthis study. L = ladder, P = pellet, CFE = cell free extract, FT = flow through, W1 = wash 1, W2 = wash 2, E = elution, B = buffer. Purification yields are as follows: wildtype M3 = 16 µg/g cells, mutant M3 = 914 µg/g cells, PCP2 = 363 µg/g cells, ul = 2.5 mg/g cells, ul = 66 µg/g cells, ulp = 434 µg/g cells, PCP3 =2.2 mg/g cells, PCP3-TE = 1.6 mg/g cells, PCP3-TE C2818A = 3.1 mg/g cells, PCP3-TE C2818 = 2.6 mg/g cells. ature Chemical Biology: doi:1.138/nchembio.2526
14 M3 FPLC purified M3* C2818A FPLC purified M3* C2818 FPLC purified β-amylose 2 kda standard ul i-ta purified ul i-ta purified ulp i-ta purified ature Chemical Biology: doi:1.138/nchembio.2526
15 i-ta purified PCP 2 i-ta purified Imidazole -TE wildtype i-ta purified -TE* C2818A i-ta purified -TE* C2818 i-ta purified upplementary Figure 13 FPLC traces of all proteins used in this study. FPLC traces were taken on a Bio-Rad GC chromatography system. M3 constructs were run on an Erich EC65 high-resolution size exclusion column (for proteins up to 65 kda). A standard of b- amylase (igma-aldrich) (2 kda size exclusion standard) is included for molecular weight reference. All other proteins were run on an Erich EC7 high-resolution size exclusion column (for proteins up to 7 kda). ature Chemical Biology: doi:1.138/nchembio.2526
16 ul Calc Da Found 3798 Da ul Calc Da Found Da ulp Calc Da Found Da (-Met) holo- Calc Da Found Da ature Chemical Biology: doi:1.138/nchembio.2526
17 PCP 2 Calc Da Found 1384 Da holo-pcp 2 apo- -TE Calc Da Found 3749 Da (-Met) holo- -TE Calc Da Found Da (-Met) -TE C2818A Calc Da Found Da (-Met) -TE C2818 Calc Da Found 3739 Da (-Met) upplementary Figure 14 UPLC-RM analysis of all proteins used in this study. Mass analysis was performed using UPLC-RM method C described in the nline Methods. ature Chemical Biology: doi:1.138/nchembio.2526
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