VAAM-Jahrestagung 2012 18.–21. März in Tübingen

90MEV003Biosynthesis of class III lantibiotics - in vitro studiesB. Krawczyk*, W.M. Müller, P. Ensle, R.D. SüssmuthTechnische Universität Berlin, Institut für Chemie , Berlin, GermanyLantibiotics represent an important class of peptide natural productssynthesized by large variety of Gramm positive bacteria. The mostcharacteristic structural feature of all lantibiotics is the presence oflanthionine (Lan) bridges, a posttranslational modification, providingstructural constraints necessary for the biological activity 1 . The ribosomalorigin and interesting biological properties turns lantibiotics into promisingtemplates for the design of new biologically active compounds. Recentlywe reported on novel class III lantibiotics named labyrinthopeptines fromActinomycetes 2 . The characteristic feature of labyrinthopeptines is aunique carbacyclic side chain linkage composed of the posttranslationallymodified triamino triacid named labionin (Lab) introduced by the LabKCenzyme 3 . In addition labyrinthopeptin A2 displays a rare activity againstneuropathic pain in mammals. In order to exploit unique features of thelabionin biosynthesis, the activity of the modifying enzyme LabKC wasreconstituted in vitro, allowing a detailed mechanistic investigation. TheLabKC enzyme, as all class III synthetases display a unique, well defineddomain arrangement in which each catalytic activity necessary for thebiosynthesis can be assigned to a specific domain (see figure). It waspossible to identify a recognition motif within the leader peptide, necessaryfor the processing by the LabKC 4 . In addition the mode of processing andthe substrate specificity were investigated providing deep insights into theactivity of class III enzymes. It was also found that the GTP preference ofLabKC is not conserved within class III lantibiotics. We believe that largestructural diversity of this class of lantibiotics and the wide spread ofhomologues enzymes in known genomes might result in discovering ofnew promising structures in the nearest future.1. Chatterjee et al., Biosynthesis and mode of action of lantibiotics. Chem. Rev. (2005) 105, 633-684.2. Meindl et al., Labyrinthopeptins: a new class of carbacyclic lantibiotics. Angew. Chem. Int. Ed. (2010) 49,1151-1154.3. Müller et. al., In vitro biosynthesis of the prepeptide of type-III lantibiotic labyrinthopeptin A2 includingformation of a C-C bond as a post-translational modification. Angew. Chem. Int. Ed. (2010) 49, 2436-2440.4. Müller et. al., Leader Peptide-Directed Processing of Labyrinthopeptin A2 Precursor Peptide by theModifying Enzyme LabKC. Biochemistry (2011) 50, 8362-8373.MEV004The Effect of MbtH-like Proteins on the Adenylation ofTyrosine in the Biosynthesis of Aminocoumarin Antibioticsand VancomycinB. Boll*, T. Taubiz, L. HeidePharmazeuisches Institut, Pharmazeutische Biologie, Tübingen, GermanyMbtH-like proteins, comprised of approximately 70 amino acids, areencoded in the biosynthetic gene clusters of non-ribosomally formedpeptides and other secondary metabolites derived from amino acids.Recently, several MbtH-like proteins have been shown to be required forthe adenylation of amino acid in non-ribosomal peptide synthesis. We nowinvestigated the role of MbtH-like proteins in the biosynthesis of theaminocoumarin antibiotics novobiocin, clorobiocin and simocyclinone D8as well as the glycopeptide antibiotic vancomycin. It could be shown thatthe tyrosine-activating enzymes CloH, SimH and Pcza361.18, involved inthe biosynthesis of clorobiocin, simocyclinone D8 and vancomycin,respectively, require the presence of MbtH-like proteins in a molar ratio of1:1. They form a heterotetramer consisting of two adenylating enzymesand two MbtH-like proteins. In contrast, NovH involved in novobiocinbiosynthesis showed activity even in the absence of MbtH-like proteins,but its activity was stimulated by the presence of MbtH-like proteins.Comparison of the active centers of CloH and NovH showed only oneamino acid to be different, i.e. L383 versus M383. A site-directedmutagenesis of this amino acid in CloH (L383M) indeed resulted in anMbtH-independent mutant. All investigated tyrosine-adenylating enzymesexhibited remarkable promiscuity for MbtH-like proteins from differentpathways and organisms. Additionally, the MbtH-like protein YbdZ fromE. coli was found to co-purify with the heterologously expressed tyrosineadenylatingenzymes and to influence their biochemical propertiesmarkedly. Therefore, a knock-out strain was created in which thecorresponding gene was deleted. This is of central importance for areliable biochemical characterization of the tyrosine-adenylating enzymes.1. Boll, B., Taubitz, T., and Heide, L. (2011) J. Biol. Chem. 286, 36281-362902. Wolpert, M., Gust, B., Kammerer, B., and Heide, L. (2007) Microbiology 153, 1413-14233. Baltz, R. H. (2011) J. Ind. Microbiol. Biotechnol. 38, 1747-1760MEV005KirCI and KirCII, the discrete acyltransferases involved inkirromycin biosynthesisE.M. Musiol*, T. Härtner, A. Kulik, W. Wohlleben, T. WeberUniversity of Tübingen, Microbiology/Biotechnology, Tübingen, Germanylarge complex of type I polyketide synthases and non-ribosomal peptidesynthetases (PKS I/NRPS complex), encoded by the genes kirAI-kirAVIand kirB [1]. The PKSs KirAI-KirAV have a “trans-AT”-architecture.These megaenzymes have no acyltransferase domains integrated into thePKS modules. In contrast, KirAVI belongs to the classical “cis-AT”-typePKS, where the ATs are part of the PKS protein. In the gene cluster ofkirromycin two separate genes, kirCI and kirCII, were identified, whichare similar to acyltransferases.To investigate the involvement of kirCI and kirCII in kirromycinbiosynthesis, mutants were generated and analyzed for kirromycinproduction. The inactivation of kirCI (kirCI) resulted in a significantreduction of kirromycin production. In kirCII the kirromycin synthesiswas completely abolished. To confirm the effects of the deletion of kirCIand kirCII, both mutants were complemented with the wild type genes. Inthe complemented strains the antibiotic production was restored to levelscomparable with the parent strain S. collinus Tü 365. These data indicatethat both genes are involved in kirromycin biosynthesis and the genekirCII is essential for the production of this antibiotic.For kirromycin assembly, a selective loading of ACPs with the buildingblocks malonyl-CoA and ethylmalonyl-CoA is required. To find outwhether KirCI and KirCII are responsible for this precursor supply and todetermine the substrate specificity of these enzymes, an in vitro ACPloading assay was carried out. Therefore KirCI, KirCII and two selectedACPs were expressed in E. coli and purified. The proteins were used in thein vitro assay and the loading of malonyl-CoA, methylmalonyl-CoA andethylmalonyl-CoA to the ACPs was monitored by autoradiography andHPLC/ESI-MS. The experiments showed that KirCI loads specificallymalonyl-CoA onto ACP4 and the second enzyme, KirCII, is the firstbiochemically characterized “trans-AT” with high specificity for ethylmalonyl-CoA and transfers this substrate to ACP5 [2]. Thus, there is a specificrecognition of the ACP of module 4 and 5 by KirCI and KirCII, respectively.To our knowledge, such interaction mechanism, where a free-standing ATproteinthat provide unusual building block, dock site-specific to the“recipient”-ACP to achieve structural diversity in polyketides was notcharacterized until now.[1]. T. Weber, K.J. Laiple, E.K. Pross, A. Textor, S. Grond, K. Welzel, S. Pelzer, A. Vente and W.Wohlleben, Chem Biol15(2008), 175-188.[2]. E.M. Musiol, T. Härtner, A. Kulik, J. Moldenhauer, J. Piel, W. Wohlleben and T. Weber, ChemBiol18(2011), 438-444.MEV006Investigation of the type II polyketide synthase from Gramnegativebacteria Photorhabdus luminescence TT01Q. Zhou*, H.B. BodeGoethe Universität Frankfurt, Molekulare Biowissenschaften, Frankfurtam Main, GermanyThe aromatic heptaketide anthraquinone (AQ-256) is produced by theentomopathogenic Gram-negative bacterium Photorhabdus lumicescenceTT01 (1). Previous studies have shown that the type II polyketide synthase(type II PKS) is responsible for the AQ-256 biosynthesis, because thetypical octaketide shunt products known from actinorhodin biosynthesiscould be identified (2). The gene cluster consists of ketosynthase (KS ),chain length factor (CLF or KS ), acyl-carrier protein (ACP), two cylases,one ketoreductase, one phosphopantetheinyl transferase (PPTase) and twoproteins with possible function as a CoA ligase (AntG) and hydrolase(AntI), respectively.In this study, we show that E. coli could be used as host for in vivoanalysis of the biosynthesis by combining two Duet vectors includingwhole or partial gene cluster. Not only the shunt products could beidentified by HPLC-MS, but also the function of the genes could beinvestigated in E. coli. Most proteins were expressed in soluble fraction in E.coli BL21 DE(3) and successfully purified. ACP could only be activated by thePPTase in company with AntG, but not by Sfp or MtaA. It looks as if thePPTase and AntG have strong interaction with each other. Site-directed mutantsof AntG were generated and their activities could be tested. Additionaldisruptions of the gene antG and antI in TT01 were also performed. Utahmycin(3) was identified in the TT01 AntI knockout mutant. The hydrolase AntI wasresponsible for heptaketide formation from octaketide. Finally, in vitroexperiments were performed leading to production of octaketide shunt productsusing the minimal PKS, KR and CYC/ARO.1. Brachmann, A. O.; Joyce, S. A.; Jenke-Kodarna, H.; Schwär, G.; Clarke, D. J.; Bode, H.B.Chembiochem2007,8(14), 1721-1728.2. McDaniel, R.; Ebert khosla, S.; Hopwood, D. A.; Khosla, C.Science1993,262(5139), 1546-1550.3. Bauer, J. D.; King, R. W.; Brady, S. F. Utahmycins A and B,Journal of NaturalProducts2010,73(5), 976-979.Kirromycin is an antibiotic produced by Streptomyces collinus Tü 365.This compound binds to the elongation factor Tu (EF-Tu) and blocksbacterial protein biosynthesis. The molecule backbone is synthesized by aBIOspektrum | Tagungsband 2012