A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis. (supplementary information)

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1 A standalone adenylation domain forms amide bonds in streptothricin biosynthesis (supplementary information) Chitose Maruyama 1, Junya Toyoda 1, Yasuo Kato 2, Miho Izumikawa 3, Motoki Takagi 3, Kazuo Shinya 4, ajime Katano 1, Tk Takashi hiutagawa 1 Yoshimitsu it amano 1 1 Department of Bioscience, Fukui Prefectural University, 411 MatsuokaKenjojima, Eiheijicho, Yoshidagun, Fukui , Japan. 2 Department of Biotechnology, Toyama Prefectural University, 518 Kurokawa, Imizushi, Toyama , Japan. 3 Japan Biological Informatics Consortium, 2432 Aomi, Kotoku, Tokyo , Japan. 4 Biomedicinal Information Research Center, ational Institute of Advanced Industrial Science and Technology, 247 Aomi, Kotoku, Tokyo 13564, Japan. 1. Supplementary Results Supplementary Figures and legends (Supplementary Figs. 1 13) Supplementary Tables (Supplementary Tables 1 6) 2. Supplementary Methods 1

2 1. Supplementary Results Supplementary Figures and legends Supplementary Figure 1. Comparison of the organization of ST biosynthetic gene clusters in Streptomyces strains. The 34kbp DA fragments containing the ST biosynthetic gene clusters from S. rochei BRC1298 and S. lavendulae BRC12789 were experimentally determined, whereas the gene organizations of S. rochei F2 (accession number, Y1293) and S. noursei JA389b (accession numbers, AJ and AJ31573) were obtained from databases. ypothetical protein, P. a X * * min. * clonat (.33 mg/ml) * (m/z = 53.3) STB (m/z = 1,15.7) (m/z = 887.6) (m/z = 759.5) (m/z = 631.4) [M] [M] [M] 1 [M] [Ma] m/z m/z [Ma] 99.6 X clonat (1 mg/ml) STB * STB (m/z = 1,15.7) (m/z = 887.6) (m/z = 759.5) (m/z = 631.4) (m/z = 53.3) min m/z m/z [M] 1 1,15.7 [Ma] 1, , 1,5 m/z

3 b [M] [M] X 1 6 * * * * m/z m/z [M] 1 [M] min m/z m/z c [M] 1 [M] * X min. * m/z m/z * * [M] 1 [M] m/z m/z d orf 5 orf 6 e f X 1 6 X min min. orf 8 g orf 11 X X min min. 3

4 h orf 12 i orf 13 X X min min. j orf 17 k orf 18 X X min min. l X * orf 19 [M] m/z min. m [M] X * m/z min. 4

5 n [M] X * m/z min. o (%) [M] 1 (%) [M] X * * min. * * m/z m/z [M] 1 [M] m/z m/z Supplementary Figure 2. PLC/ESIMS analysis of the metabolites produced by Streptomyces strains. (a) STs (clonat, a mixture of,,,, and STB) used as reference standards. (b) S. rochei BRC1298. (c) S. lividans TK23 harboring pj446srcluster7. (d) S. lividans TK23 harboring pj446srcluster7 orf5. (e) S. lividans TK23 harboring pj446srcluster7 orf6. (f) S. lividans TK23 harboring pj446srcluster7 orf8. (g) S. lividans TK23 harboring pj446srcluster7 orf11. (h) S. lividans TK23 harboring pj446srcluster7 orf12. (i) S. lividans TK23 harboring pj446srcluster7 orf13. (j) S. lividans TK23 harboring pj446srcluster7 orf17. (k) S. lividans TK23 harboring pj446srcluster7 orf18. (l) S. lividans TK23 harboring pj446srcluster7 orf19. (m) S. lavendulae BRC (n) S. lividans TK23 harboring pj446slcluster12. (o) S. lividans TK23 harboring pj446slcluster12 and plae9orf19. Extracted ion chromatograms for (m/z 53.3), (m/z 631.4), (m/z 759.5), ST C(m/z 887.6), and STB (m/z 1,15.7) are shown. An asterisk denotes mass spectra of selected compounds. 5

6 RF5 1 MESSESSFLEPFFDTARDDPDRPAVVDGLVLTYGTFAAWVARVAGAVEPRTAEPQPPVGVVASARDVAAILGVLA 78 SttA 1 MVDGLVIGYGTLAAWALAVAGAVAPRTVAPQPPVGIVVTARDVAAILGVLA ARDVAAILG VLA 54 psa 1 MESSASSFLEPFFDTARDDPDRPAVVDGLVLTYGTFAAWVARVAGAVEPRTAEPQPPVGVVASARDVAAILGVLA 78 RF19 1 MTLPSVLDPFFAAARSPERPAVIDGMTVGYGQLASWAATVADLVATRTPGPPPVAVVTSVRDIAAILGVLA 76 PacU 1 MSLTLVERFLRGLEVSPDRPAVRIGSDVVTYREADTASRWAGSLMADGSPRTVGVLAGKSTTSYVGLLAVLY 73 ovl 1 MAKDAPEYVTRILAEATLDGARPVVRWRDTVITGTQLDRSVRRVVTALREAGVARDAVAVLTQVSPWMLIVRYAALVGASVVYI 9 CouL 1 MARDGPEYVTRILDEAARDGARPVVRWRDTVITGTELRSVRRVATALREAGVARDAVAILTQVSPWMLVVRYAALLGASVVYI 9 RF5 79 AGRAYVPLEAPEPRLEGILRRVGCREAVATAETGWQPPVEKVIRPRWTPAAGPSASPEGASARLPRPEDPAYVLF 155 SttA 55 TGRAYVPLEAGPEARLESVLALAGCREAVATAETGWQPPVETVIRPRWTQTPVTTVLQGTLPESGARPEDPAYMLF 131 psa 79 AGRAYVPLEAPEPRLEGILRRVGCREAVATAETGWQPPVEKVIRPRWTPAAGPSASPEGASARLPRPEDTAYVLF 155 RF19 77 AGRGYVVVPGEDAGRAGDALRALGCRELIATADTGPLPAVDRVLRPRWTPSRAPVRGGRPVGADDPAYVLF 148 PacU 74 AGATVVPLLPFPAALTRWMIKLAGVETLIVDERGLAVLPEITDRDTSFRVLDTGGFADRFPTIPVTSPVISAPVPVAPSDQAYILFVAPSDQAYILF 161 ovl 91 TGAGIVTELPVATRVRMLREAGASVLVFDESAQLAETVDETVRDKLVLCGLGPASGTVSVDGRPVDDVSVDFTPEAPELAMVLY 179 CouL 91 TGAGTVTDLPVTTRVRMMREAGASVLVFDERAQLAETIRETVPDKLVLCGLGPASGTVTADGRPVEDVAVEFPAETPELAMVLY 179 RF5 156 TSGSTGEPKGVVVPRAPAAVVPPLRGLYGIGDGESVLFGVGGDTSLEEILPTLTGGGTLVLDDGAREGFARVAKEQQVSVAVLPTG 244 SttA 132 TSGSTGEPKGVVVPRAPAAVVPLLRDLYGIGPEETVLFGAGGDTSLEEILPALTGGATLVIDDAAPEGFARVAEEQQVSVAILPTG 22 psa 156 TSGSTGEPKAVVVPRAPAAVVPPLRALYGIGDGESVLFGVGGDTSLEEILPTLTGGGTLVLDDGAREGFARVAEEQQVSVAVLPTG 244 RF TSGSTGAPTAVAVPRALAVLPLIGRYGAGPTVSLFTRADGDTSLEEILPPLLTGGLVVLDGDAEADLDRVLTDEVTLILPVD 237 PacU 162 TSGSTGVPKGVPISGSFTYFELQDRRCDFGPDDVISQTFDLFDCGIVVFSAWGAGATVQTVPPSAYRDLPAFLAEGITVWYSTPS 251 ovl 18 TSGTTGQPKGVCRSFGSWAAALRGAAYPRPVFLTMTAVSQTVAMIVDTVLAAGGSVLLRERFDPADFLRDVGERVTETFMGVA 264 CouL 18 TSGTTGQPKGVCKPFGAWATVVGLAGQPRPRQTYLAMTAVSTVGMVVDIALAAGGSVLLREKFDPTDFLRDVVTRVTDTFMGVP 266 RF5 245 FWDSLTGDLLGARLPASLRTVVIGGEAVRADMLERWRVRGAEDVRLLTYGSTETALVTAVQLAGPGAPVLPETGG 324 SttA 221 FWSSLTGDLLQGARLPESLRTVVIGGEAVRADMLERWRRLPGTDGVRLLTYGSTETALVTAAQLAGPGAPALPGTGL 3 psa 245 FWDSLTGDLLGARLPASLRTVVIGGEAVRADMLERWRVRGAEDVRLLTYGSTETALVTAVQLAGPGAPVLPETGG 324 RF YWIYTGLLETGRKLPGSVETVVIGGEAVRPDMLERWRLGADGVRLLTYGSTETALVTSAVLAGPGAETTVAGSADAEQPSGGAS 326 PacU 252 GIWMAREMGGLDKGALDGLRWSFFAGEALRCQDAADWQAAAPGSKVELYGPTELTITVCRRWSGEESERRGVGTV 329 ovl 265 QLYAILGPDARTADLSSLRVLYLGCPASPERLREAAALLPGVLAQSYGSTEAGRITVLRAADERPELLATVGRA 341 CouL 267 QLYAILPDVRTTDLSSLQLVYVGCPASPERLREAVTVFPGVLWQSYGSTETGRIAMLREDDDPELLATVGRP 343 RF5 325 DLPIGLPLTVGQLVDESGELYVSGPGLALGYDDPGATAARFTERDGTRWYRTGDLVGEAAGGLLVFRGRSDQV 4 SttA 31 DLPIGPLPVRQRVDDRGELSIAGPGLALGYGPGATSARFRERDGTRWYRTGDLVTEAPGGILVFRGRSDQV Q 376 psa 325 DLPIGLPLTVGQLVDESGELYVSGPGLALGYDDPGATAARFTERDGTRWYRTGDLVGEAAGGLLVFRGRSDQV 4 RF FADGVPIGRPLPSVRQAVVPRPDAPDGPGELFVSGPQLATGYLDDPARTAARFVEADLGGGPARWYRTGDLVEAADGTLVFGRLDQV 416 PacU 33 PIGQVEADFRLVTEQGQRSSVEGELWVTGPQMATGYLDPADEEGRFVDLDGRRWYRTGDRVRARPDGELAYIGRLDSQV 41 ovl 342 VPGVTIAIRDPETGDLPVGEIGEVVVGPEVMAGYVADPETARVIRDGWVTGDFGSVDERGYVRLFGRMREVV 417 CouL 344 MPGVTIAIRDPQTGRDLPVEIGEVVVSPMAGYIGDPEGTTRVVRDGWVTGDMGSVDERGYVRLFGRMEMV 419 RF5 41 KIRGFRVDLLDVEALIGRCEGVSSVAAARVERAGSSLAAFFVPLPDFAPGEVA 454 SttA 377 KIRGFRVDLLDVEALIGRCEGVAAVAAARVDRTEGVLAAFFVALPREPDEIA 43 psa 41 KIRGFRVDLLDVEALIGRCEGVSSVAAARVERAGSSLAAFFVPLPDFAPGEVA 454 RF KIRGRVDLLDVEEAIGRLPGVTAVAAAPSKGETALVAFVVLAPEATAPDDAPASRDGGPAPATTAPDSTGADGTSADVTGPA 56 PacU 411 KVGWRVELAEVDVVRECDGVRGAVTVTRTALGTELVVFYTGDEVPET 46 ovl 418 KVQDTRVSPTEVEKVLVGCPGVVDACVYGRGPDLIEELAAVVLGTEGAPSFDT 472 CouL 42 KVQDTRVSPTEVEKVLVGCPGVVDACVYGRRPDLIEELAAVVLSTEDAPSFAA 474 RF5 455 AAIRERLTRTAAPLVPGLIVPVDALERTTGKVDRATRDRLGTVEAAR 55 SttA 431 AGIREGLARTAPPLIPSLIVPVEALEQTRTGKVDRATRDRLDATAAPR 481 psa 455 AAIRERLTRTAAPLVPGLIVPVDALERTTGKVDRATRDRLGTVEAAR 55 RF19 57 GSATTAPDPAALRARLRATLPLVPDRIVAVPELATRTGKVDRATTRDRYL 559 PacU 461 VLAAGVRAVLPDSILPRRYVLAAFPLSRKVDRIALRERAAGLAG 57 ovl 473 LRDVARAMTPTAPIRFVRWRRFPITGKVRLRVREVSAEARGDSPDVLVDR 527 CouL 475 LRDVAQTMTPTAPVRFVRWRQFPITGKTDRLRIREVSAEARGEGPDVLVDR 529 Supplementary Figure 3. Alignment of standalone Adomains. Identical and similar aminoacid residues are shown by dark and lightshaded boxes, respectively. The numbers on the left and right are the aminoacid residues. The alignment was derived using the Clustal W program ( 6

7 a X [M] min Streptothrisamine (1, 2) m/z b 1 spectrum c 13 C spectrum 7

8 d bserved data Reported data , Streptothrisamine (1) 1Decarbamoyl12carbamoyl streptothrisamine (2) 12Carbamoyl e DQFCSY spectrum 8

9 CTMBC spectrum 2 2 Streptothrisamine (1) 1Decarbamoyl12carbamoyl streptothrisamine (2) Supplementary Figure 4. Elucidation of the chemical structures of streptothrisamine (1) and 1 decarbamoyl12carbamoyl streptothrisamine (2). (a) The culture broth of S. lividans TK23 harboring pj446 SRcluster7 orf18 was analyzed by PLC/ESIMS. Extracted ion chromatograms for streptothrisamines (1 and 2, m/z 375.2), (m/z 53.3), (m/z 631.4), (m/z 759.5), and (m/z 887.6) are shown. The mass spectra of streptosamines (1 and 2) are shown in the righthand panel. 1 MR (b) and 13 CMR (c) spectra of the mixture of streptothrisamine (1) and 1decarbamoyl12carbamoyl streptothrisamine (2) were recorded at 6 and 15 Mz, respectively, using the Varian MR system 6 B CL. (d) MR data of streptothrisamine (1) and1 decarbamoyl12carbamoyl c b streptothrisamine s e (2) from the present pese study sudyweree compared to those of and 12 carbamoyl 1.(e) Twodimensional experiments, double quantum Filteredcorrelation spectroscopy (DQF CSY) and constant timeheteronuclear multi bond connectivity (CTMBC), were performed at ambient temperature, and the chemical structures of streptothrisamine (1) and 1decarbamoyl12carbamoyl streptothrisamine (2) were elucidated. 9

10 a 1 and 13 C MR spectra bserved data Reported data , , , Carbamoyl Carbamoyl b 2DMR spectra bserved data Reported data 2 :CSY,TCSY 2 : MBC 2 12Carbamoyl Supplementary Figure 5. Elucidation of the chemical structures of and 12carbamoyl. The sample isolated from the culture broth of S. lividans TK23 harboring pj446srcluster7 was analyzed by MR. (a) 1 and 13 CMR spectra were recorded at 6 and 15 Mz, respectively, using the Varian MR system 6 B CL. MR data of and 12carbamoyl from the present study were compared to those of and 12carbamoyl 1.(b) Twodimensional experiments, CSY, totally correlated spectroscopy (TCSY), and heteronuclear multi bond connectivity (MBC) were performed at ambient temperature, and the chemical structures were elucidated. 1

11 a ( kda ) lane 1 ; Molecular weight marker lane 2 ; rrf 5 ( 54 kda ) lane 3 ; rrf 18 ( 65 kda ) lane 4 ; rrf 19 ( 59 kda ) 25 2 b Protein size marker min., 67 kda mau 1.4 min., 142 kda min., 32 kda min., 29 kda min., 12.4 kda min. Purified rrf 5 rrf 5 mau (11.23 min., 6 kda) min. Purified rrf 18 rrf 18 mau (1.68 min., 11 kda) kda min. Purified rrf 19 mau 16 8 rrf 19 (1.79 min., kda) min. Reaction mixture containing rrf 5, rrf 18, and rrf 19 rrf 18 (1.74 min., 11 kda) mau 12 rrf 19 (1.74 min., kda) 6 rrf 5 (11.22 min., 6 kda) min kda 142 kda rrf 18 (11 kda) rrf 5 (6 kda) rrf 19 ( kda) 67 kda 32 kda 12.4 kda min.

12 c rrf 5 rrf 19 L Lysine LLysine LGlysine LAlanine LSerine LThreonine LCysteine LValine LLeucine LIsoleucine LMethionine LProlineL LPhenylalanine LTyrosine LTryptophan LAspartic acid LGlutamic acid LAsparagine LGlutamine Listidine LArginine none 5 Relative activity (%) 5 Relative activity (%) d ATP (nm) 8 6 rrf 5 ( U mg 1 ) min. rrf 19 (19 3 U mg 1 ) Supplementary Figure 6. Enzymatic characterizations ti of rrf 5, rrf 18, and rrf 19. (a) The purified recombinant enzymes rrf 5, rrf 18, and rrf 19 were subjected to SDSPAGE. Proteins were stained with CBB R25. (b)the relative molecular masses of rrf 5, rrf 18, and rrf 19 were estimated by gelfiltration chromatography (SunSec Diol, 4 m, mm, Chromaik Technologies, saka, Japan). In addition, the enzyme reaction mixture containing rrf 5, rrf 18, and rrf 19 was analyzed. Glutamate dehydrogenase (29 kda), lactate dehydrogenase (142 kda), enolase (67 kda), myokinase (32 kda), and cytochrome C (12.4 kda) (riental Yeast) were used as the standard molecular masses.(c) Relative adenylation activities were determined on L lysine and proteinogenic amino acids. Each value represents the mean of three experiments. (d) Specific activities of rrf 5 and rrf 19 were determined. In this assays, 1 µg/ml purified enzymes were employed. ne unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 nmol ATP per min at 3 C. 12

13 rrf 5 rrf 18 rrf 19 1 L Lysine a b c d 7 ( X 1 ) min. Lys 7mer (6) Lys 6mer (5) Lys 5mer (4) Lys 4mer (3) rrf 5 rrf 18 rrf 19 1 L Lysine e f g h 7 ( X 1 ) min. Lys 7mer (6) Lys L 6mer (5) Lys 5mer (4) Lys 4mer (3) rrf 5 rrf 18 rrf 19 1 L Lysine 7 ( X 1 ) i j k l min. Lys 7mer (6) Lys 6mer (5) Lys 5mer (4) Lys 4mer (3) 13

14 rrf 5 rrf 18 rrf 19 1 L Lysine 7 ( X 1 ) m n o p min. rrf 5 rrf 18 rrf 19 1 L Lysine 7 ( X 1 ) q r s min. rrf 5 rrf 19 3 L Lysine Lys 7mer (6) Lys 6mer (5) Lys 5mer (4) Lys 4mer (3) 6 Lys 7mer (6) 7 ( X 1 ) Lys y 7mer (6) 4 Lys y 6mer (5) Lys 6mer (5) Lys 5mer (4) Lys 4mer (3) min. Lys 5mer (4) Lys 4mer (3) rrf 5 rrf 18 rrf 19 (233V) 1 L Lysine Lsine t 7 ( X 1 ) u Lys 7mer (6) Lys 6mer (5) Lys 5mer (4) Lys 4mer (3) min. 14

15 v w [M] 1 [M] [Ma] x [M] [Ma] m/z m/z m/z y z [M] [M] [Ma] [Ma] m/z m/z Supplementary Figure 7. rrf 5, rrf 18, and/or rrf 19 reactions with streptosamine (13), L lysine (12), and/or (1). (as) Enzymatic reactions were analyzed by PLC/ESIMS. Extracted ion chromatograms for (m/z 53.3), (m/z 631.4), (m/z 759.5), (m/z 887.6), L lysine oligopeptide consisting of four residues (L Lys 4mer) (3, m/z 531.4), L Lys 5mer (4, m/z 659.5), L Lys 6mer (5, m/z 787.6), and L Lys 7mer (6, m/z 915.7) are shown. The mass spectra of L Lys 4mer (t) and L Lys 5mer (u), produced by the reaction with rrf 5, rrf 18, and rrf 19 and with streptosamine (1) andl lysine, were analyzed by PLC/ESIMS. The mass spectra of 3 (v) and4 (w) produced by the reaction with rrf 5, rrf 18, and rrf 19 and with 1 and L lysine as the substrates, were analyzed by PLC/ESIMS. The mass spectra of 4 (x), 5 (y), and 6 (z) produced by the reaction with rrf 5, rrf 18, and rrf 19 and with L lysine as the sole substrate, were analyzed by PLC/ESIMS. a b c ( 1 7 ) [M] [M] [M] min. 2 3 m/z Supplementary Figure 8. Investigation of the linkage pattern of lysine residues in the enzymatically synthesized lysine oligopeptide. (a) ε(2,4dinitrophenyl) lysine ( ε DP Lys), (b) (2,4 dinitrophenyl) lysine y ( DP Lys), y), (c) hydrolysate y of the DPprotected lysine y oligopeptide gpp (7mer) (6) produced by rrf 5, rrf 18, and rrf 19. Extracted ion chromatograms for DP Lys (m/z 313.1) are shown. The mass spectra of DP Lys are shown in the righthand panel. ε DP Lys and DP Lys used as the reference standards were prepared by the method previously reported 2. 15

16 rrf 19 L Lysine a b c d 8 ( X 1 ) 2 1 rrf 5 L Lysine * * * * min. e f g h STA STB 8 ( X 1 ) 2 STA STB 1 * * * * min. Supplementary Figure 9. rrf 5 or rrf 19 reactions with, and/or L lysine. (ah) Enzyme reactions were analyzed by PLC/ESIMS. Extracted ion chromatograms for (m/z 759.5), (m/z 887.6), STB (m/z 1,15.7), and STA (m/z 1,143.8) are shown. An asterisk denotes impurities containing the substrates ( and ). Supplementary Figure 1. Substrate specificities for rrf 5 and rrf 19. Relative adenylation activities were determined on L lysine and L lysine analogs. Each value is represented as the mean of three experiments. Error bars are not shown, because relative standard deviations of less than 5% were commonly calculated. 16

17 a ( 1 7 ) rrf 5 ( 1 3 ) ( 1 4 ) , deconvolution min. 1, 1,5 2, 2,5 m/z. 53,5 54, 54,5 55, 55,5 m/z 56, b ( 1 7 ) rrf19 ( 1 3 ) ( 1 4 ) 59, deconvolution min.. 1, 1,5 2, 2,5 m/z. 58, 58,5 59, 59,5 6, 6,5 m/z c d e f ( 1 4 ).3 ( 1 4 ).2 ( 1 4 ) , 1,5 2, 2,5 m/z. 1, 1,5 2, 2,5 m/z. 1, 1,5 2, 2,5 m/z Supplementary Figure 11. Molecular masses of rrf 5 and rrf 19 after the reactions. (a) rrf5was incubated with L lysine. (b) rrf 19 was incubated with L lysine. (c) rrf 18 was incubated with L lysine. The enzyme reactions were analyzed by PLC/QTF MS using a reversedphase column. The chromatograms, mass spectra, and deconvoluted mass spectra of enzymes are shown in the lefthand panels, the middle panels, and the righthand hand panels, respectively. (d) Mass spectra of rrf 18 incubated with rrf 19 and L lysine. L (e) Mass spectra of rrf 18 incubated with rrf 5 and L lysine. (f) Mass spectra of rrf 18 incubated with rrf 5, rrf 19, and L lysine. 17

18 a rrf 5 rrf 18 rrf 19 1 L omolysine 7 ( X 1 ) hlys 5mer (12) hlys 4mer (11) hlys 3mer (1) SThD (9) SThE (8) SThF (7) min. rrf 5 rrf 18 rrf 19 1 L omolysine 7 ( X 1 ) hlys 5mer (12) hlys 4mer (11) hlys 3mer (1) SThD (9) SThE (8) SThF (7) min. 18

19 b SThF (7) [M] m/z 1 and 13 C MR spectra 2DMR spectra : CSY, TCSY :MBC 19

20 c SThE (8) [M] m/z Chemical Formula: C Exact Mass: Molecular Weight: m/z: (.%), (3.1%), (7.2%), (3.7%) Elemental Analysis: C, 49.23;, 7.65;, 21.26;, d SThD (9) [M] [Ma] m/z e f g hlys 3mer (1) [M] hlys 4mer (11) [M] [Ma] 69.4 hlys 5mer (12) [M] [Ma] m/z m/z m/z Supplementary Figure 12. rrf 5, rrf 18, and rrf 19 reactions with streptothrisamine (1) and L homolysine. (a) Enzyme reactions were analyzed by PLC/ESIMS. Extracted ion chromatograms for SThF (7, m/z 517.3), SThE (8, m/z 659.4), SThD (9, m/z 81.4), L homolysine oligopeptide consisting of three residues ( hlys 3mer) (1, m/z 445.2), L hlys 4mer (11, m/z 587.4), and L hlys 5mer (12, m/z 729.5) are shown. (b) 7 purified from the enzyme reaction was analyzed by MR. 1 and 13 CMR spectra were recorded at 6 and 15 Mz, respectively, using the Varian MR system 6 B CL. The mass spectra of 8 (c), 9 (d), 1 (e), 11 (f), and 12 (g) are shown. 2

21 8 Survival rate ( % ) Streptothrisamine (1) L lysine oligopeptides (6 residues) (5) SThF (7) ygromycin B (Positive control) 1 1 Concentration (μm) Supplementary Figure 13. Cytotoxic activities of the synthesized compounds. Cytotoxic activity against human cervical carcinoma ela cells was determined by a colorimetric assay, using 2(2 methoxy4nitrophenyl)3(4nitrophenyl)5(2,4disulfophenyl)2tetrazolium monosodium salt (WST8). Cells were cultured in DMEM medium (Sigma) supplemented with 1% (v/v) fetal bovine serum (Life Technologies), penicillin ( U ml 1 ), and streptomycin ( g ml 1 )at37 C ina humidified incubator under a 5% C 2 atmosphere. The 384well plates were seeded with aliquots of 2 μl medium containing cells per well, and were incubated overnight before being treated with compounds at various concentrations for 48 h. Plates were incubated for 1 h at 37 C afterthe addition of 2 μl of WST8 reagent solution (Cell Counting Kit; Dojindo) per well. The absorption of the formed formazan dye was measured at 45 nm. 21

22 Supplementary Tables Supplementary Table C (15 Mz) and 1 (6 Mz) MR data a for streptothrisamine (1) and 1decarbamoyl12carbamoyl streptothrisamine (2) Streptothrisamine (1) 1Decarbamoyl12carbamoly streptothrisamine (2) o. C (multiplicity, J = z) C (multiplicity, J = z) (d, 14.6) (d, 14.6) (dd, 14.6, 1.8) (dd, 14.4, 1.8) (m) (m) (dd, 14.7, 5.6) (dd, 14.7, 5.6) 3.42 (dd, 14.7, 1.2) 3.42 (dd, 14.7, 1.2) (d, 9.6) (d, 9.5) (dd, 9.6, 3.) (m) (t, 3.) (t, 2.9) (m) (d, 2.9) (t, 6.2) (t, 6.1) (d, 6.2) (d, 6.1) a MR spectra were obtained with the Varian MR system 6 B CL in D 2, and the solvent peak was used as an internal standard ( 48. ppm). 22

23 Supplementary Table C (15 Mz) and 1 (6 Mz) MR a data for and 12carbamoyl 12Carbamoyl o. C (multiplicity, J in z) C (multiplicity, J in z) (d, 13.8) (d, 13.8) (dd, 13.8, 2.4) (dd, 13.8, 2.4) (ddd, 6.7,2.4, 1.2) (ddd, 6.7,2.4, 1.2) (dd, 14.5, 6.7) (dd, 14.5, 6.7) 3.35 (dd, 14.5, 1.2) 3.35 (dd, 14.5, 1.2) (d, 9.7) (d, 1.) (dd, 9.7, 2.9) (dd, 1., 2.6) (t, 2.9) (t, 2.6) (dd, 3.2, 2.9) (dd, 3., 2.6) (dt, 3.2, 6.8) (dt, 6.5, 3.)) (d, 6.8) (d, 6.5) (dd, 14.7, 4.4) (dd, 14.7, 4.4) 2.38 (dd, 14.7, 8.5) 2.38 (dd, 14.7, 8.5) ( 3.25 (m) ( 3.25 (m) (m) (m) 1.53 (m) 1.53 (m) (m) (m) (t, 7.) (t, 7.) a MR spectra were obtained with the Varian MR system 6 B CL in D 2, and the solvent peak was used as an internal standard ( 4.8 ppm). 23

24 Supplementary Table 3. Alignment of the 1 aminoacid residues conferring substrate specificity in the Adomain RPSs a GrsA Residue position according to GrsA numbering D A W T I A A I C K Substrate D/LPhe RF 5 D T E V V G T L V K Lys RF 19 D T E 233 V G T L V K Lys SttA b D T E I V G T L V K Lys psa c Cmn c D T E V V G T L V K D T E D V G T M V K a sequence data derived from: GrsA (PID no. g39369) b substrate specificity was not investigated, but it was suggested to be lysine 3 c substrates were determined to be lysine y 4 Lys Lys 24

25 Supplementary Table C (15 Mz) and 1 (6 Mz) MR a data for SThF (7) SThF (7) o. C (multiplicity, J in z) (d, 12.) (dd, 12., 3.2) a (dd, 14.7, 5.6) 3.41 (dd, 14.7, ) (d, 9.7) (dd, 9.7, 3.3) (t, 3.3) a (dt, 7., 4.1) (d, 7.) (dd, 16.7, 4.4) 2.67 (dd, 16.7, 8.2) (m) (m) (m) (m) (t, 7.7) a MR spectra were obtained with the Varian MR system 6 B CL in D 2, and the solvent peak was used as an internal standard d ( 4.8 ppm). a verlapped with a solvent peak. 25

26 Supplementary Table 5. Antibiotic activities in STrelated compounds MIC ( M) Microorganisms SThF (7) Streptothrisamine (1) L Lysine L Lysine oligopeptide (six residues) (5) Escherichia coli W > 1, > 1, > 1, Bacillus subtilis BRC < 1 3 > 1, > 1, 125 Staphylococcus aureus AB > 1, > 1, > 1, Saccharomyces cerevisiae S288C > 1, > 1, > 1, Saccharomyces pombe L > 1, > 1, > 1, Yeasts and bacteria were grown for 2 d at 3 C and for 1 d at 37 C, respectively. 26

27 Supplementary Table 6. ligonucleotides used in this study ligonucleotides Sequences Experiments SATF 5 GACGCSGARGCSATCGARGSSCTSGA 3 SAT gene amprification SATR 5 GTTSTYGTTSGTSACYTCSAGCCA 3 SAT gene amprification rf 51P1 5 GTGGCACGGGTGGCGGGTGCCGTGGAGCC GCGCACGGCGATTCCGGGGATCCGTCGACC 3 Inactivation of the orf 5 gene rf 52P2 P1 site 5 CGAGTGCCCGGCGCGCTCCACCCGTGCGG CCGCGACCGATGTAGGCTGGAGCTGCTTCG 3 Inactivation of the orf 5 gene P2 site rf 61P1 5 CTCCAGCTGGGCTTCCTCCTCTACTGGGTGC TGTACGACATTCCGGGGATCCGTCGACC 3 Inactivation of the orf 6 gene rf 62P2 P1 site 5 AGTGACGTACACCGTCAGGACGGTGTCGCC GACCACGGTTGTAGGCTGGAGCTGCTTCG 3 Inactivation of the orf 6 gene rf 81P1 P2 site 5 GGCGTGTTCTCGCTGGGGGCCGACAACCCG CCGGCCGTCATTCCGGGGATCCGTCGACC 3 Inactivation of the orf 8 gene rf 82P2 P1 site 5 CAGCTCGATGCCCCAGAAGACGTCGTGGACC CCGGCCCGTGTAGGCTGGAGCTGCTTCG 3 Inactivation of the orf 8 gene rf 111P1 P2 site 5 GGGCTCCCGGTGCGGTGGGAGGGCACGGCC Inactivation of the orf 11 gene TCGTCGCTGATTCCGGGGATCCGTCGACC 3 rf 112P2 P1 site 5 GGACACGGGCTGGCGGATCACCAGCGACCG GTGCGCGATTGTAGGCTGGAGCTGCTTCG 3 Inactivation of the orf 11 gene rf 121P1 P2 site 5 GTGGTCGTCCGCGGCCACGAGTTCGACCAAC AGCGCATCATTCCGGGGATCCGTCGACC 3 Inactivation of the orf 12 gene rf 122P2 P1 site 5 CAGCACGTCGCCCTGGTCGGCGACGACCTCC CGCATGCCTGTAGGCTGGAGCTGCTTCG 3 Inactivation of the orf 12 gene P2 site rf 131P1 5 ACGGTGCTGGGCATGCTCGCGGCACTGAAGG CGGGGGCCATTCCGGGGATCCGTCGACC 3 Inactivation of the orf 13 gene rf 132P2 P1 site 5 TGCGCTCGCCCGTAGCCCGGCCGCCGAGGG GAGGAACTCTGTAGGCTGGAGCTGCTTCG 3 Inactivation of the orf 13 gene rf 171P1 P2 site 5 TGGGACCTGCCCAAGCTCTTCGGCGACCGCG GTCTGAACATTCCGGGGATCCGTCGACC 3 Inactivation of the orf 17 gene rf 172P2 P1 site 5 CAGCACCACCGGGGTGCCGGTGTGCCCGCC GACCTCGCCTGTAGGCTGGAGCTGCTTCG 3 Inactivation of the orf 17 gene P2 site f181p1 rf 181P1 5 CTCACGCCGGAGGCGGACCTGTCGGCCCCG Inactivation of the orf f18 gene CTGCTCGACATTCCGGGGATCCGTCGACC 3 rf 182P2 P1 site 5 CAGCACGTCGTCGTCGGTCGTGGCGATCACT TCGATGAGTGTAGGCTGGAGCTGCTTCG 3 Inactivation of the orf 18 gene P2 site rf 191P1 5 GCCCTCGGCTGCCGGGAGCTGATCGCCACC GCCGACACCATTCCGGGGATCCGTCGACC 3 Inactivation of the orf 19 gene rf 192P2 P1 site 5 GGTGGCGCTGCCGTGGGCAGGGCCGGTGAC GTCGTGGGCTGTAGGCTGGAGCTGCTTCG 3 Inactivation of the orf 19 gene P2 site rrf 5F 5 GGGGGATCCGAGTCCTCCGAATCCTCCTTT 3 Construction of rrf 5 rrf 5R 5 ACCAAGCTTTCATCGGGCGGCCTCCACGGT 3 Construction of rrf 5 rrf 13F 5 GGGGGATCCACCGCCGTGGACGCTCCCGAC 3 Construction of rrf 13 rrf 13R 5 ACCAAGCTTCTACCGGGGCAGACCCGCCGC 3 Construction of rrf 13 rrf 18F rrf 18R 5 GGAATTCCATATGACCGCCCCCACCCGCCCC 3 5 ACCAAGCTTTCAGTGGTGGTGGTGGTGGTG GTGGTGGCTGTCCGCCGCCCCGGCCGCACC 3 Construction of rrf 18 Construction of rrf 18 rrf 19F 5 GGGGGATCCACCCTGCCCAGCGTGCTCGAT 3 Construction of rrf 19 rrf 19R 5 ACCAAGCTTTCACAGGTACCGGTCCCGGGT 3 Construction of rrf 19 27

28 2. Supplementary Methods eterologous expression of the S. lavendulae BRC12789 ST biosynthetic gene cluster with the orf 19 gene. pqe3rf19 (see METDS section) was digested with Bam I and ind III, and the orf 19 gene fragment was ligated with an expression vector plae9 (carrying the neomycinresistant gene aph II) 5. The resulting plasmid (plae9orf19) was introduced into S. lividans TK23 harboring pj446slcluster12 (see METDS section). The resulting transformant was cultured in S1.3 medium (see METDS section) containing apramycin (5 μg/ml) and neomycin (5 μg/ml) for 8 d at 25 C. The supernatant of the culture broth was analyzed by PLC/ESIMS (Esquire 4, Bruker) using a reversedphase column (see METDS section). Constructions of rrf 5, rrf 13, rrf 18, rrf 19, and rrf 19 (233V). The following three sets of PCR primers were designed and used to amplify the orf 5, orf 13, and orf 19 genes: rrf 5F and rrf 5R for construction of rrf 5; rrf 13F and rrf 13R for rrf 13; rrf 19F and rrf 19R for rrf 19 (Supplementary Table 6). PCRs were carried out under standard conditions. Amplified DA fragments (orf 5, 1.5 kbp; orf 13, 2. kbp; orf 19, 1.7 kbp) were ligated into pqe3 (Qiagen). After confirmation of their sequence, the resulting plasmids, pqe3rf 5, pqe3rf 13, and pqe3rf 19 were introduced into E. coli M15(pREP4) for expression as terminally 6istagged fusion proteins. Cells were harvested from 5 ml culture broth by centrifugation at 6, g for 15 min, resuspended in 5 ml Buffer A (5 mm TrisCl, 2% glycerol, 3 mm acl, and 2 mm DTT, p 8.) containing 1 mm imidazole, and sonicated on ice. Insoluble material was removed by centrifugation at 12, g for 15 min. The supernatant was run on a 1ml nickelnitriloacetic acid (ita) Sepharose column (Qiagen) that had been preequilibrated with 5 ml Buffer A containing 1 mm imidazole. The column was washed with 5 ml Buffer A containing 2 mm imidazole, and recombinant enzymes were eluted with 1 ml Buffer A containing 25 mm imidazole and used for in vitro enzyme reactions. rrf 18F and rrf 18R PCR primers were designed and used to amplify the orf 18 gene (Supplementary Table 6). The PCR product was ligated with expression vector psa81 (a gift from Prof. Kobayashi of the University of Tsukuba, Tsukuba, Japan). The resulting plasmid was introduced into S. lividans TK23 using a standard procedure 6 and the transformant was inoculated into S1.3 medium containing thiostreptone (2 g/ml). After growth for 3 d at 3 C, the cells were collected from 5 ml culture broth and sonicated in Buffer A. Cterminal 8istagged RF 18 (rrf 18) 28

29 was purified by the same procedure used for rrf 5, rrf 13, and rrf 19. To construct rrf 18 (Q288A), the following primers were designed and used to change Gln288 to Ala: 5 TTGATCGTCGTCCACGCGCTCGCGTTCGACATG 3 (q288af) and 5 CATGTCGAACGCGAGCGCGTGGACGACGATCAA3 (q288ar). ucleotides at the site of mutation are shown in bold. The Quickchange XL sitedirected mutagenesis kit was used to generate rrf 18 (Q288A). To construct rrf 19 (233V), the following primers were designed and used to change Asn233 to Val: 5 GAGGTCACCCTGATCGTCCTGCCCGTCGACTAC3 (n233vf) and 5 GTAGTCGACGGGCAGGACGATCAGGGTGACCTC3 (n233vr). ucleotides at the site of mutation are shown in bold. The Quickchange XL sitedirected mutagenesis kit was used to generate rrf 19 (233V), using pqe3rf 19 as the PCR template. After confirmation of the sequence of the resulting plasmid, pqe3rf 19 (233V), it was introduced into E. coli M15(pREP4). rrf 19 (233V) was purified by the same procedure used for rrf 5, rrf 13, and rrf 19. Assay of Adomain aminoacid selectivity. The activation of proteinogenic amino acids and amino acids by rrf 5, rrf 13, and rrf 19 was investigated using standard ATP 32 PP i exchange assays followed by liquidscintillation counting 7. All assays were carried out under linear conditions. ne unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 nmol ATP per min at 3 C. Protein concentrations were determined using a BioRad Protein Assay Kit (BioRad). Bovine serum albumin was used as the standard protein. L homolysine (95%) and D/L aminobutyric acid (95%) were purchased from Watanabe Chemical Industries (iroshima, Japan). L alanine (97%) was purchased from Wako Chemical (Tokyo, Japan). Purification of streptothrisamines (1 and 2). The S. lividans TK23 transformant harboring pj446srcluster7 orf18, in which the orf 18 gene is inactivated by inframe deletion (see METDS section), was cultured in S1.3 medium (see METDS section) containing apramycin (5 μg/ml) for 8 d at 25 C. The culture broth (1 L) was centrifuged, and the supernatant obtained was mixed with 1 L chloroform. After vigorous shaking, the aqueous layer from centrifugation was mixed with Diaion P2 resin of 25 g (Wako) and the suspension was filtered. The filtrate was loaded on a Dowex 5WX2 column ( 2 mesh, 4 form, 4. 1 cm) and the column was washed with 2 ml water. The sample was eluted stepwise with 2 ml of,, 1.5, and 2. M ammonium bicarbonate. The streptothrisamine fractions eluted with and 29

30 M ammonium bicarbonate were combined and applied to an activatedcarbon column (2. 1 cm) that had been equilibrated with water. The column was washed with water, and the streptosamine fraction was eluted with 5% acetone in.1 Cl. After removal of the organic solvent, the aqueous layer was lyophilized to give white powder. This sample was dissolved in a small volume of water, and fractionated by preparative PLC using a reversedphase column (Sunniest RPAQUA, 5 µm, 25 1 mm, Chromaik Technologies) at 3 C, at a flow rate of 7 ml/min, and with 4% acetonitrile/water in.5% nheptafluorobutyric acid and.5% formic acid. Fractions were collected and monitored by an ultraviolet (UV) detector at 21 nm. The fraction containing the purified streptothrisamines (1 and 2) was lyophilized to give white powder (approximately 45 mg) and used for MR analysis to determine its chemical structure. The purified streptothrisamine (1) sample containing 1decarbamoyl12carbamoyl streptothrisamine (2) (1/2 ratio of 5:1) was also employed for in vitro enzyme reactions (see METDS section). Purification of. The S. lividans TK23 transformant harboring pj446srcluster7 was cultured in S1.3 medium (see METDS section) containing apramycin (5 μg/ml) for 8 d at 25 C. The culture broth (4 L) was centrifuged, and the supernatant obtained was mixed with 4 L chloroform. After vigorous shaking, the aqueous layer from centrifugation was loaded on a Dowex 5WX2 column ( 2 mesh, 4 form, cm). The column was washed with 8 ml of.5 M ammonium bicarbonate in 3% acetonitrile, and the sample was eluted stepwise with 2 ml of.25,, and M ammonium bicarbonate in 3% acetonitrile. The streptosamine fractions eluted with and M ammonium bicarbonate were combined and lyophilized after the organic solvent was evaporated. The dried sample was dissolved in a small volume of water, and insoluble materials were removed by centrifugation. The watersoluble fraction was applied to an SCX column (5 g, Varian). The column was washed with water, and the fraction was eluted with a linear gradient of 4 M acl. The fraction containing was applied to an activatedcarbon column (4. 1 cm). The column was washed with water, and the fraction was eluted with 5% acetone in.1 Cl. After removal of the organic solvent, the aqueous layer was lyophilized to give a yellow powder. This dried sample was dissolved in a small volume of water and then fractionated by preparative PLC using a reversedphase column (Sunniest RPAQUA, 5 m, 25 2 mm, Chromaik Technologies) at 4 C, at a flow rate of 18.9 ml/min, and with 2% acetonitrile/water in.1% nheptafluorobutyric acid. Fractions were collected and monitored by a UV detector at 21 nm. The fraction containing the purified was 3

31 lyophilized to give a white powder (approximately 38 mg) used for MR analysis to determine its chemical structure. The purified was also employed for in vitro enzyme reactions (see METDS section). MR spectroscopy. 1 and 13 CMR spectra were recorded at 6 and 15 Mz, respectively, using the Varian MR system 6 B CL. ne and twodimensional experiments (double quantum Filteredcorrelation spectroscopy (DQFCSY) and constant timeheteronuclear multi bond connectivity (CTMBC)) were performed at ambient temperature. The samples were dissolved in D 2. Isolation of L lysine from STs. A solution of STs (15 mg clonat, see METDS section) dissolved in 2 ml of 4 Cl was heated at 121 C for 2 min. The hydrolysate was concentrated to dryness in vacuo. The sample was dissolved in 1 ml water and lyophilized. The dried sample was dissolved in 1 ml water and loaded on an SCX column (5 g, Varian). The sample was eluted stepwise with 2 ml of.3,.6,.125,.25,, and M ammonium bicarbonate. The L lysine fractions eluted with and M ammonium bicarbonate were combined and lyophilized. The dried sample was dissolved in 2 ml water, passed through an SAX column ( mg, Varian) and C18 column ( mg, Varian), and then lyophilized. The sample was further purified by chromatography on a cellulose column (2. 1 cm, Advantec C) with a solvent of Bu/pyridine/Ac/ 2 /tertbu (15:1:3:12:4) 8. The fraction containing L lysine was lyophilized and the purity (95%) was confirmed by ESIMS (Esquire 4, Bruker). To confirm the chemical structure, 1 and 13 CMR spectra were compared to those of the chemically synthesized ᴅ/L lysine described below. Purified L lysine was used as the substrate for the in vitro enzyme reaction. Synthesis of D/L lysine. Dioxane solution (15 ml) of ditbutyl dicarbonate (11 mmol) was added to an icecold solution of 1amino4butanol (1 mmol) and triethylamine (15 mmol) in 66% aqueous dioxane (45 ml), and the mixture was stirred at room temperature for 4 h. After evaporation of the solvent in vacuo, the residue was distributed between ethyl acetate and 1% citric acid solution, and the reaction product was extracted with ethyl acetate three times. The combined organic phase was subsequently washed with 5% sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. After evaporation, 1.85 g (97.9% yield) of tbutyl(4hydroxybutyl)carbamate was obtained as a colorless oil. 1 MR (CDCl 3 ) 4.68 (br, 1), 3.66 (t, 2, J = 6 z), 3.15 (brd, 2), 1.94 (br, 1), (m, 4), 1.44 (s, 9); 13 CMR (CDCl 3 ) 156.1, 62.3, 4.2, 31

32 29.6, 28.4, 27.3, S 3 pyridine (4 mmol) was carefully added under an argon atmosphere to an icecold solution of tbutyl(4hydroxybutyl)carbamate (9.52 mmol) and triethylamine (4 mmol) in anhydrous DMS (25 ml), and the mixture was stirred for 5 h at the same temperature. Ice was added to the mixture to quench the reaction and the product was extracted with ethyl acetate three times. The combined organic phase was subsequently washed with 1% citric acid solution, 5% sodium bicarbonate solution, and brine, and was dried over anhydrous sodium sulfate. After evaporation of the solvent, 1.54 g crude tbutyl(4oxobutyl)carbamate was obtained as a pale yellow oil and used for the next step without further purification. A mixture of tbutyl(4oxobutyl)carbamate (8.18 mmol), malonic acid (1 mmol), ammonium acetate (2 mmol), and 2 ml of 95% Et was refluxed overnight 9. After evaporating the solvent, 1 ml acetone was added to the residue and the insoluble materials were filteredoff. To the filtrate was added 2 ml of 6 Cl in dioxane and the mixture was stirred overnight at room temperature. After evaporation, the remaining Cl in the mixture was further removed by azeotropic evaporation with toluene/me. The residue was dissolved into water and applied to a column of Dowex 5W ( form; ml: equilibrated with.2 M sodium acetate buffer (p 3.) containing 1 M acl). After the column had been washed with 3 ml each of the same acetate buffer and distilled water, the product was eluted with 3 ml of 2 M 4. After evaporation of the solvent, the residue was dissolved into 2 ml water and the p of the mixture was adjusted to 6. by 1 M 2 S 4. Me was added to the neutralized solution and evaporated to dryness, affording.223 g (11.2% yield, 2 steps) of ᴅ/L lysine monosulfate as pale yellow crystals. 1 MR (D 2 ) 3.9 (m, 1), 3.34 (t, 2, J = 7.2 z), 2.94 (dd, 1, J = 4.6, 17.8 z), 2.84 (dd, 1, J = 9.6, 17.8 z), (m, 1), (m, 2), (m, 1); 13 CMR (D 2 ) 177.6, 48.7, 38.9, 38., 29., 22.8; MS (ESI) m/z (M ). Isolation of enzymatically synthesized L lysine oligopeptides. A reaction mixture (2 ml) consisting of mm TrisCl (p 9.), 5 mm MgCl 2, 5 mm ATP, 5 mm 2mercaptoethanol, 2 mm L lysine, and 2 μg/ml enzymes (rrf 5, rrf 18, and rrf 19) was incubated at 3 C for 1 h. The synthesized L lysine oligopeptides (four to seven residues) were isolated from the reaction mixture with 12molybdosilicate ([SiMo 12 4 ] 4 ) in a similar manner to the isolation of polyllysine 1 ; 2 ml solution containing.2 mm a 2 Si 3, 1 mm a 2 Mo 4, and.4 M Cl, in which the yellow [SiMo 12 4 ] was formed, was added to the reaction mixture to obtain a paleyellow 32

33 precipitate, which consisted of the polyelectrolyte salts of protonated L lysine oligopeptides with [SiMo 12 4 ] 4. The precipitate was collected, and dissolved in a small volume of.1 M ammonia solution, in which L lysine oligopeptides were deprotonated and [SiMo 12 4 ] was hydrolyzed. Then the isolated L lysine oligopeptides were fractionated by preparative PLC using a reversedphase column (Sunniest RPAQUA, 5 µm, 25 1 mm, Chromaik Technologies) at 3 C, at a flow rate of 7 ml/min, and with a gradient of acetonitrile/water in.1% nheptafluorobutyric acid run over 25 min (25 4% acetonitrile for 2 min, 9% acetonitrile for.1 min, and 9% acetonitrile for 4.9 min). Fractions were collected and monitored by a UV detector at 21 nm. The fractions containing L lysine oligopeptides (four residues), L lysine oligopeptides (five residues), and L lysine oligopeptides (six residues), were lyophilized and used for ATP 32 PP i exchange assays (see METDS section). 33

34 References 1. Ji, Z., Wang, M., Zhang, J., Wei, S. & Wu, W. Two new members of streptothricin class antibiotics from Streptomyces qinlingensis sp. nov. J. Antibiot. 6, (27). 2. ishikawa, M. & gawa, K. Distribution of microbes producing antimicrobial polyllysine polymers in soil microflora determined by a novel method Appl. Environ. Microbiol. 68, (22). 3. FernandezMoreno, M.A., Vallin, C. & Malpartida, F. Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation. J. Bacteriol. 179, (1997). 4. Grammel,., et al. A βlysine adenylating enzyme and a βlysine binding protein involved in poly βlysine chain assembly in nourseothricin synthesis in Streptomyces noursei. Eur. J. Biochem. 269, (22). 5. Yamanaka, K., et al. Development of a recombinant epsilonpolyllysine synthetase expression system to perform mutational analysis. J. Biosci. Bioeng. 111, (211). 6. Kieser, T. et al. Practical Streptomyces Genetics (The John Innes Foundation, orwich, UK, 2). 7. Lee, S.G. & Lipmann, F. Tyrocidine synthetase system. Methods Enzymol. 43, (1975). 8. Sawada, Y., akashima, S. & Taniyama,. Biosynthesis of streptothricin. VI. Mechanisms of lysine and its peptide formation. Chem. Pharm. Bull. 25, (1977). 9. Lázár, L., Martinek, T., Bernáth, G., & Fülöp, F. A simple synthesis of βalkylsubstituted amino acids, Synth. Commun. 28, (1998). 1. Katano,., Maruyama, C., & amano, Y. Detection of biopolymer polyllysine with molybdosilicate anion for screening of the synthetic enzymes. Int. J. Polym. Anal. Charact. 16, (211). 34