Proceedings book download - 5Z.com

More documents

Recommendations

Info

Results and DiscussionSite-directed mutagenesis was performed on the pTUC202 plasmid (containing the MccJ25biosynthesis cluster) by using the QuickChange XL Site-Directed Mutagenesis Kit (AgilentTechnologies). The plasmid carrying mutations was transformed into a producing strain,which was then cultured in M63 medium to assess the production of mature MccJ25 in theculture supernatant by LC-MS detection.Sequence analysis of McjB enabled to hypothesize that McjB could be a proteaseresponsible for the cleavage of the precursor McjA [2]. Site-directed mutagenesis allowedto show that McjB is actually a cysteine protease, where Cys150, His182 and Glu186 arecrucial residues for its activity, probably constituting a catalytic triad. The involvement ofCys150 of McjB in the maturation process has been confirmed in vitro by analyzing theproduction of MccJ25 from recombinant McjA in the presence of either recombinant McjBor McjB[C150A], and recombinant McjC, ATP and Mg 2+ .The homologue of McjC, the asparagine synthetase, catalyses the activation of thecarboxyl group via an acyl-AMP intermediate and subsequent formation of the amide bond.Sequence analysis of McjC revealed conserved residues involved in the binding of ATPand Mg 2+ [2]. Site-directed mutagenesis of these residues (Ser199, Asp203 and Asp302)confirmed that they are crucial for McjC activity. It thus provides evidence that McjC isresponsible for the activation of the side chain of Glu8 of MccJ25, which is a pre-requiredstep for the macrolactam ring formation.Therefore, the study shows that the biosynthesis of MccJ25 requires the involvementof two enzymes: one is a cysteine protease (McjB), which cleaves the precursor McjA,while the other (McjC) activates the substrate possibly by formation of an acyl-AMPintermediate. Prior to the macrolactam ring closure, the folding of the precursor must occurso that the C-terminal tail can be properly trapped within it (Figure 1B). However the orderof these different steps remains to be determined.Our work provides insights into the biosynthesis of lasso peptides. Such knowledgewould probably have important biotechnological impacts, as the lasso structure incombination with its maturation enzymes could be used to generate peptides endowed withnovel bioactivities. The understanding of the mechanisms underlying the formation of thelasso structure is therefore a major step towards the engineering of lasso peptides.AcknowledgmentsThis work was supported by the ANR project n BLAN-NT09-692063. We acknowledge the massspectrometry platform at the MNHN for access to the ESI-Qq-TOF spectrometer as well as thebacteriology facility.References1. Rebuffat, S., Blond, A., Destoumieux-Garzón, D., Goulard, C., Peduzzi, J. Curr. Protein Pept. Sci.5, 383-391 (2004).2. Duquesne, S., Destoumieux-Garzón, D., Zirah, S., Goulard, C., Peduzzi, J., Rebuffat, S. Chem. Biol.14, 793-803 (2007).3. Knappe, T.A., Linne, U., Zirah, S., Rebuffat, S., Xie, X., Marahiel, M.A. J. Am. Chem. Soc. 130,11446-11454 (2008).4. Salomón, R.A., Farías, R.N. J. Bacteriol. 174, 7428-7435 (1992).5. Rosengren, K.J., Clark, R.J., Daly, N.L., Goransson, U., Jones, A., Craik, D.J. J. Am. Chem. Soc.125, 12464-12474 (2003).397
Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Enterocins L50A and L50B from Enterococcus durans A5-11:Conformational and Antibacterial StudiesSéverine Zirah 1 , Christophe Goulard 1 , Rémi Ducasse 1 ,Michèle Dalgalarrondo 2 , Jean Peduzzi 1 , Jean-Marc Chobert 2 ,Thomas Haertlé 2 , and Sylvie Rebuffat 11Muséum National d’Histoire Naturelle, Centre National de la Recherche Scientifique,Laboratoire Molécules de Communication et Adaptation des Microorganismes,FRE 3206 CNRS-MNHN, 75005 Paris, France. 2 Institut National de la RechercheAgronomique, UR 1268, Biopolymères Interactions Assemblages, équipeFonctions et Interactions des Protéines Laitières, Nantes, FranceIntroductionBacteriocins produced by lactic acid bacteria are ribosomally-synthesized peptides thatshow potent antibacterial activity against certain Gram-positive bacteria and in particularfood pathogens such as Listeria. They present a strong interest as potential foodpreservatives. Class IIb bacteriocins consist of two different unmodified peptides for whichoptimal antibacterial activity requires the presence of both peptides in about equal amounts[1,2], while class IId includes leaderless peptides (class II2 in the enterocin classification)[3], synthesized without an N-terminal leader sequence.Two antibacterial peptides secreted by Enterococcus durans A5-11 isolated fromMongolian airag (traditional fermented mare's milk) [4] were purified from culturesupernatants and characterized in terms of amino-acid sequences, conformations,antibacterial activities, and potential synergy.Results and DiscussionIsolation. Enterococcus durans A5-11 isolated from Mongolian airag was grown in M17medium at 37°C overnight. The supernatant was subjected to SP-Sepharosecation-exchange chromatography. The active fraction (elution in 0.6 M NaCl) was saltedout by solid-phase extraction on a C 18 cartridge. Enterocins were further purified byreversed-phase high-performance liquid chromatography on an Uptisphere C 4 column.Peptide sequences. The purified peptides were identified as enterocins L50A and L50Bisolated previously from Enterococcus faecium [5]. Despite 72% of sequence identity, thetwo peptides showed different sensibility to proteases, L50A and L50B being resistant andsensible to trypsin, respectively.L50A: f-MGAIAKLVAK FGMPIVKKYY KQIMQFIGEG WAINKIIEWI KKHIL50B: f-MGAIAKLVTK FGMPLIKKFY KQIMQFIGQG WTIDQIEKWL KRHTable 1. Minimal inhibitory concentrations of L50A and L50BEntL50AMIC (nM)EntL50 A/BEntL50B75/25 50/50 25/75Lactobacillus brevis F1.114 156 312 156 156 312Enterococcus faecium 156 312 nd 156 ndLactobacillus sakei sp.sakei 500 nd 250 ndListeria ivanovii sp. ivanovii N29 312 625 312 312 625Lactobacillius plantarum 625 312 nd 625 nd398
Page 2 and 3:
Peptides 2010Tales of PeptidesProce
Page 4 and 5:
Peptides 2010Tales of PeptidesProce
Page 8 and 9:
IntroductionWe had the distinct hon
Page 10:
Scientific programIn planning the 3
Page 13 and 14:
31 st EUROPEAN PEPTIDE SYMPOSIUMSep
Page 19 and 20:
published by John Wiley & Sons as a
Page 21 and 22:
The Josef Rudinger AwardThis award
Page 23 and 24:
Young Investigators' SymposiumThe y
Page 25 and 26:
xxiv
Page 27 and 28:
Design of Peptidyl-Inhibitors for G
Page 29 and 30:
Synthetic Antifreeze Glycopeptide A
Page 31 and 32:
Synthesis and Oxidative Folding of
Page 33 and 34:
Convergent Syntheses of Huprp106-12
Page 35 and 36:
Bactericidal Activity of Small Beta
Page 37 and 38:
Bradykinin Analogues Acylated on Th
Page 39 and 40:
Antimicrobial Activity of Small 3-(
Page 41 and 42:
Interaction of Curcumin with A-Synu
Page 43 and 44:
Fluorescent and Luminescent Fusion
Page 45 and 46:
Cellular Expression of the Human An
Page 47:
2D IR Spectroscopy of Oligopeptides
Page 50 and 51:
large, non-redundant subset of prot
Page 52 and 53:
The aberrantly glucosylated peptide
Page 54 and 55:
Table 1. Proline cis/trans ratios o
Page 56 and 57:
Table 1. Yields, UV-vis absorption
Page 58 and 59:
Table 1. Purity of the targeted tri
Page 60 and 61:
processes are blocked. Interestingl
Page 62 and 63:
(tb4 n ,1 m )MTII(tb1 n ,4 m )MTIIF
Page 64 and 65:
Fig. 2. a) Part of the 400 MHz HR-M
Page 66 and 67:
Once printed, the amino acid partic
Page 68 and 69:
A) PSA, few seconds73B) PSA, 45 min
Page 70 and 71:
short β strands. In contrast, nume
Page 72 and 73:
neg. control Cs9-Rhd MM-218Fig. 2.
Page 74 and 75:
Table 1. The general structure of t
Page 76 and 77:
% ID in the liver 1 h p.i.100908070
Page 78 and 79:
Peptidyl phosphoranes were first re
Page 80 and 81:
obtained from the same sardines and
Page 82 and 83:
MccJ25 variants were produced by si
Page 84 and 85:
Fig. 2. Proposed signaling mechanis
Page 86 and 87:
Table 1. mCD4-HS12 antiviral activi
Page 89 and 90:
Proceedings of the 31 st European P
Page 91 and 92:
Page 93 and 94:
Page 95 and 96:
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
RT: 0,00 - 5,9936000034000032000030
Page 111 and 112:
Page 113 and 114:
Page 115 and 116:
Page 117 and 118:
Page 119 and 120:
Page 121 and 122:
Page 123 and 124:
Page 125 and 126:
Page 127 and 128:
Page 129 and 130:
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Page 137 and 138:
Page 139 and 140:
Page 141 and 142:
Page 143 and 144:
Page 145 and 146:
Page 147 and 148:
Page 149 and 150:
Page 151 and 152:
Page 153 and 154:
Page 155 and 156:
Page 157 and 158:
Page 159 and 160:
Page 161 and 162:
Page 163 and 164:
Page 165 and 166:
Page 167 and 168:
Page 169 and 170:
Page 171 and 172:
Page 173 and 174:
Page 175 and 176:
Page 177 and 178:
Page 179 and 180:
Page 181 and 182:
Page 183 and 184:
Page 185 and 186:
Page 187 and 188:
Page 189 and 190:
Page 191 and 192:
Page 193 and 194:
Page 195 and 196:
Page 197 and 198:
Page 199 and 200:
Page 201 and 202:
Page 203 and 204:
Page 205 and 206:
Page 207 and 208:
Page 209 and 210:
Page 211 and 212:
Page 213 and 214:
Page 215 and 216:
Page 217 and 218:
Page 219 and 220:
Page 221 and 222:
Page 223 and 224:
Page 225 and 226:
Page 227 and 228:
Page 229 and 230:
Page 231 and 232:
Page 233 and 234:
Page 235 and 236:
Page 237 and 238:
Page 239 and 240:
Page 241 and 242:
Page 243 and 244:
Page 245 and 246:
Page 247 and 248:
Page 249 and 250:
Page 251 and 252:
Page 253 and 254:
Page 255 and 256:
Page 257 and 258:
Page 259 and 260:
Page 261 and 262:
Page 263 and 264:
Page 265 and 266:
Page 267 and 268:
Page 269 and 270:
Page 271 and 272:
Page 273 and 274:
Page 275 and 276:
Page 277 and 278:
Page 279 and 280:
Page 281 and 282:
Page 283 and 284:
Page 285 and 286:
Page 287 and 288:
Page 289 and 290:
Page 291 and 292:
Page 293 and 294:
Page 295 and 296:
Page 297 and 298:
Page 299 and 300:
Page 301 and 302:
Page 303 and 304:
Page 305 and 306:
Page 307 and 308:
Page 309 and 310:
Page 311 and 312:
Page 313 and 314:
Page 315 and 316:
Page 317 and 318:
Page 319 and 320:
Page 321 and 322:
Page 323 and 324:
Page 325 and 326:
Page 327 and 328:
Page 329 and 330:
Page 331 and 332:
Page 333 and 334:
Page 335 and 336:
Page 337 and 338:
Page 339 and 340:
Page 341 and 342:
Page 343 and 344:
Page 345 and 346:
Page 347 and 348:
Page 349 and 350:
Page 351 and 352:
Page 353 and 354:
Page 355 and 356:
Page 357 and 358:
Page 359 and 360:
Page 361 and 362:
Page 363 and 364:
Page 365 and 366:
Page 367 and 368:
Page 369 and 370:
Page 371 and 372:
Page 373 and 374:
Page 375 and 376:
Page 377 and 378:
Page 379 and 380:
Page 381 and 382:
Page 383 and 384:
Page 385 and 386:
Page 387 and 388:
Page 389 and 390:
Page 391 and 392:
Page 393 and 394: Proceedings of the 31 st European P
Page 443: Proceedings of the 31 st European P
Page 495 and 496:
Page 497 and 498:
Page 499 and 500:
Page 501 and 502:
Page 503 and 504:
Page 505 and 506:
Page 507 and 508:
Page 509 and 510:
Page 511 and 512:
Page 513 and 514:
Page 515 and 516:
Page 517 and 518:
Page 519 and 520:
Page 521 and 522:
Page 523 and 524:
Page 525 and 526:
Page 527 and 528:
Page 529 and 530:
Page 531 and 532:
Page 533 and 534:
Page 535 and 536:
Page 537 and 538:
Page 539 and 540:
Page 541 and 542:
Page 543 and 544:
Page 545 and 546:
Page 547 and 548:
Page 549 and 550:
Page 551 and 552:
Page 553 and 554:
Page 555 and 556:
Page 557 and 558:
Page 559 and 560:
Page 561 and 562:
Page 563 and 564:
Page 565 and 566:
Page 567 and 568:
Page 569 and 570:
Page 571 and 572:
Page 573 and 574:
Page 575 and 576:
Page 577 and 578:
Page 579 and 580:
Page 581 and 582:
Page 583 and 584:
Page 585 and 586:
Page 587 and 588:
Page 589 and 590:
Page 591 and 592:
Page 593 and 594:
Page 595 and 596:
Page 597 and 598:
Page 599 and 600:
Page 601 and 602:
Page 603 and 604:
Page 605 and 606:
Page 607 and 608:
Page 609 and 610:
Page 611 and 612:
Page 613 and 614:
Page 615 and 616:
Page 617 and 618:
Page 619 and 620:
Page 621 and 622:
Page 623 and 624:
Page 625 and 626:
Page 627 and 628:
Page 629 and 630:
Page 631 and 632:
Page 633 and 634:
Page 635 and 636:
Page 637 and 638:
Page 639 and 640:
Page 641 and 642:
Page 643 and 644:
Page 645 and 646:
Page 647 and 648:
Page 649 and 650:
Page 651 and 652:
Page 653 and 654:
Page 655 and 656:
Page 657 and 658:
Page 659 and 660:
Page 661 and 662:
Page 663 and 664:
Page 665 and 666:
Page 667 and 668:
Page 669 and 670:
Page 671 and 672:
Page 673 and 674:
Page 675 and 676:
Page 677 and 678:
Page 679 and 680:
Subject index(S)-2-(1-adamantyl)gly
Page 681 and 682:
chip 18chiral triazine condensing r
Page 683 and 684:
heat stress 348helical conformation
Page 685 and 686:
O-acyl isopeptide method 112obesity
Page 687 and 688:
SH2-ligands 210short collagen-relat
Page 689 and 690:
Author indexAboye, Teshome Leta 142
Page 691 and 692:
Chi, Hongfang 480Chiche, Laurent 6C
Page 693 and 694:
Geronikaki, Athina 444Gessmann, Ren
Page 695 and 696:
Kostova, Kalina 528Kotzia, Georgia
Page 697 and 698:
Nagata, Koji 162Nagayev, Igor Yu. 5
Page 699 and 700:
Salvarese, Nicola 518Sammet, Benedi
Page 701 and 702:
Vauquelin, Georges 138Veiga, Ana Sa
show all

Proceedings book download - 5Z.com

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?