FU Berlin Digitale Dissertation
Hanka Symmank : Functional and structural characterization of bacterial peptide synthetases Peptide engineeriengby combinatorial manipulation of surfactin synthetase Funktionelle und strukturelle Charakterisierung bakterieller Peptidsynthetasen
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Abstract Due to their modular organization nonribosomal peptide synthetases (NRPS) offer a most promising target for the directed engineering of new bioactive compounds. However, the ignoring of interdomain interactions led to many failures in former module swapping experiments. Therefore this work was focussed on the systematic evaluation of possible recombination sites on their impact on distinct enzymatic functions to provide the required instruments for the biocombinatorial reorganization of NRPS. Each module is at least composed of three functional domains responsible for the adenylation (A), thioester formation (T) and condensation (C) of the peptide amino acids. The A-domains determine the substrate specificity and thus play a key role in this process. This was taken into account by a detailed analysis of two members from the adenylate forming family. At first the autonomously acting acetyl-CoA synthetase from Lysobacter sp. ATCC 53042 was chosen, which accepts not only an unusually broad range of carboxylic acid substrates, but also shows considerable differences between the forward and reverse reactions of the two-step acyl-CoA synthesis in dependence on the provided ligand combinations. Secondly, the first module Srf-M1 of the surfactin synthetase from B. subtilis ATCC 21332 was characterized which was unable to recognize and activate other amino acids than its major substrate L-glutamic acid. It possesses two protease-sensitive regions corresponding exactly to the previously found hinge region dividing the A-domain into two subdomains as well as the linker area between the C- and the adjacent A-domain. These flexible regions represent the most obvious reasons for the failure of the extensive crystallization attempts. In view of the main objective, the next step comprised the systematic rearrangement of distinct surfactin synthetase fragments and modules and the examination of the effects of the different recombination strategies. As model enzymes both monomodular hybrids between the coding regions of the L-valine and the L-leucine activating modules Srf-M4 and Srf-M7 and bimodular hybrid enzymes between the L-glutamic acid and the D-leucine activating modules Srf-M1 and Srf-M3 from the trimodular surfactin synthetase SrfA-A were generated in which fusion sites from all domain types A, T and C were used. It was demonstrated that only recombinations within the highly conserved His-motif resulted in a completely active peptide synthetase. By contrast, only partial catalytic competence was preserved by other manipulations like fusions within the hinge region between both A-subdomains or within the highly conserved T-motif. After deletion of a complete module unit the recombinant surfactin synthetase from B. subtilis R13CDM produced in vivo only a lipohexapeptide according to its new programming. Because of the reduced toxicity against erythrocytes it is conceivable that this derivative will become a suitable alternative for the wild type surfactin.

Table of Contents Download the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title Titelblatt Abkürzungsverzeichnis 1 Einleitung und Problemstellung 2 Grundlagen und Theorie 2. 1 Modulare Organisation der nichtribosomalen Peptidsynthetasen 2. 2 Der Multienzym Thiotemplate Mechanismus 2. 3 Die funktionellen Domänen der nichtribosomalen Peptidsynthetasen 2. 4 Die Surfactin-Synthetase 3 Material 3. 1 Mikroorganismen 3. 2 Plasmide 3. 3 Primer für die Polymerasekettenreaktion 3. 4 Chemikalien, Enzyme, Kits und Größenmarker 3. 5 Apparative Ausstattung und sonstige Materialien 3. 6 Medien, Agarplatten und Antibiotika 3. 7 Puffer und Lösungen 4 Methoden 4. 1 Aufbewahrung und Anzucht von Bakterien 4. 2 Methoden zur Isolierung, Analyse, Amplifikation und Rekombination von DNA 4. 3 Methoden zur Isolierung und Charakterisierung von Proteinen 4. 4 In vitro-Bestimmung enzymatischer Aktivitäten 4. 5 In vivo-Biosynthese von Surfactin und -analoga 4. 6 Synthese von 3-Hydroxytetradecanoyl-Coenzym A 4. 7 Kristallisationsansätze 5 Ergebnisse 5. 1 Enzymatische Charakterisierung der Acetyl-CoA-Synthetase von Lysobacter sp. ATCC 53042 5. 2 Enzymatische und strukturelle Charakterisierung der A-Domäne des Glutaminsäure-aktivierenden Startmoduls der Surfactin-Synthetase Srf-M1 5. 4 In vivo-Rekombination von Surfactin-Synthetase-Genen in B. subtilis und Charakterisierung der resultierenden Deletions- und Austauschmutanten der Surfactin-Synthetase 6 Diskussion 7 Literatur 8 Anhang 9 Veröffentlichungen und Präsentationen

More Information:
Online available:	http://www.diss.fu-berlin.de/2002/75/indexe.html
Language of PhDThesis:	german
Keywords:	peptide synthetases, peptide engineering, novel surfactin, surfactin synthetase
DNB-Sachgruppe:	30 Chemie
Date of disputation:	19-Apr-2002
PhDThesis from:	Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee:	Prof. Dr. Wolfram Saenger
Second Referee:	Prof. Dr. Joachim Vater
Contact (Author):	hsymmank@web.de
Contact (Advisor):	fbern@bpc.uni-frankfurt.de
Date created:	10-May-2002
Date available:	13-May-2002

 

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