4’-Phosphopantethein Transfer in Bacillus subtilis und Pseudomonas aeruginosa sowie Grundlagen zur gerichteten Proteinevolution von Adenylierungsdomänen der Nichtribosomalen Peptidsynthetasen

Carrier Proteine (CP) werden in unterschiedlichen prokaryontischen und eukaryontischen Synthesesystemen als Transporteinheiten für die Syntheseintermediate eingesetzt. Im Primärmetabolismus dienen sie zum Transport von Acyleinheiten (Fettsäuresynthese) und D-Ala (Modifikation der Zellwand). Im Sekun...

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Bibliographic Details
Main Author: Finking, Robert
Contributors: Marahiel, Mohamed A. (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2003
Online Access:PDF Full Text
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Table of Contents: Carrier Proteins (CPs) function as transport units for intermediates in a number of different prokaryotic and eukaryotic machineries. In primary metabolism, CP are used to transport acyl units (fatty acid synthesis) or D-Ala (modification of the bacterial cell wall) whereas in secondary metabolism they serve as transporters for aminoacyl/peptidyl/aryl-units (nonribosomal peptide synthetases, NRPS) and ketoacyl units (polyketide synthases). Intermediates are generally bound as thioesters to the 4’-phosphopantetheine cofactor (4’PP) of the CP. The 4’PP cofactor is transferred posttranslationally from Coenzyme A to a conserved serine residue of the CP, catalyzed by so-called 4’PP-transferases (PPTases). E. coli harbors two PPTases. The Acyl Carrier Protein Synthase (AcpS) modifies solely the CP of primary metabolism, whereas EntD recognizes only the CPs of secondary metabolism. This work revealed that the two PPTases of B. subtilis, AcpS and Sfp, exhibit overlapping selectivity: while AcpS recognizes only the CPs of primary metabolism, Sfp modifies all CPs present, albeit with an obvious preference for those of secondary metabolism. Only one open reading frame encoding a PPTase, PcpS, was found in the genome of P. aeruginosa. According to its physical characteristics, PcpS is a PPTase of secondary metabolism. Biochemical characterization of PcpS revealed, however, that it, like Sfp, modifies all types of CPs but was evolutionary optimized for the modification of CPs of primary metabolism. Deletion of the corresponding gene and RNA-interference showed that this PPTase is essential for P. aeruginosa. Further characterization of the CP-PPTase recognition threw light on the mode of interaction of the two proteins. The concept of inhibition of adenylation domains (A-domains) of NRPS, which are responsible for the selection of the amino acid substrate, by aminoacyl-sulfamoyl adenylate analogues was successfully transferred to an A-domain of primary metabolism. Modification of these inhibitors with a linker enables binding to a matrix. In general, the linker had no influence on the inhibitory properties of these molecules and may allow their usage in directed protein evolution of A-domains. In addition, a screening system for the evolution of A-domains was developed that relies on the luminescence of the enzyme Luciferase which can be followed in vitro. This system allows screening of an A-domain library for changed selectivity towards the substrate of Luciferase, namely Luciferin.