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Papers by Martin Hahn
Proceedings of the National Academy of Sciences of the United States of America, Jan 2, 2004
Nonribosomal peptide synthetases (NRPSs) catalyze the formation of structurally diverse and biolo... more Nonribosomal peptide synthetases (NRPSs) catalyze the formation of structurally diverse and biologically important peptides. Given their modular organization, NRPSs provide an enormous potential for biocombinatorial approaches to generate novel bioactive compounds. Crucial for the exploitation of this potential is a profound knowledge of the intermolecular communication between partner NRPSs. The overall goal of this study was to understand the basis of protein-protein communication that facilitates the selective interaction in these multienzyme complexes. On this account, we studied the relevance of short regions at the termini of the NRPSs tyrocidine (Tyc) synthetases TycA, TycB, and TycC, constituting the Tyc biosynthetic template. In vitro and in vivo investigations of C-terminal deletion mutants of the initiation module TycA provided evidence for the existence and impact of short communication-mediating (COM) domains. Their decisive role in protein-protein recognition was subse...
ChemBioChem, 2006
Assembly of bioactive natural compounds through the action of nonribosomal peptide synthetases (N... more Assembly of bioactive natural compounds through the action of nonribosomal peptide synthetases (NRPSs) relies on the specific interplay of modules and domains along these multiple mega-enzymes. As the C termini of several bacterial NRPSs often harbor epimerization (E) domains that generate D-amino acids, these seem to facilitate the ordered intermolecular enzymatic interaction and the directed transfer of intermediates. To elucidate this bifunctional role, E domains in recombinant bimodular proteins derived from the tyrocidine synthetase B were investigated. By utilizing sequent tryptic proteolysis and HPLC Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), we could directly interrogate and determine the formation of intermediates attached to the TycB(3)-PCP domain of wild-type TycB(2-3) and to the E domain exchange enzyme TycB(2-3)-ATCAT/E(tycA). In addition, the two proteins and a version of TycB(2-3) fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with TycB(1) to corroborate E domain impact on intermodular NRPS interaction. Significant functional differences between the C-terminal aminoacyl- and peptidyl-E domains were observed in terms of in trans interaction and misinitiation. E domains originating from elongation modules (peptidyl-E domains) seem to be optimized for regulation of the progression of peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas E domains of initiation modules (aminoacyl-E domains) impair upstream condensation and cause misinitiation. The selection of E domains is therefore decisive for successful application in biocombinatorial engineering of nonribosomal peptides.
The interaction between enzymes of a nonribosomal peptide synthetase (NRPS) complex relies on the... more The interaction between enzymes of a nonribosomal peptide synthetase (NRPS) complex relies on the interplay of compatible sets of donor and acceptor communication-mediating (COM) domains. Hence, these domains are essential for the formation of a defined biosynthetic template, thereby directing the synthesis of a specific peptide product. Without the selectivity provided by different sets of COM domains, NRPSs should form random biosynthetic templates, which would ultimately lead to combinatorial peptide synthesis. This study aimed to exploit this inherent combinatorial potential of COM domains. Based on sequence alignments between COM domains, the crosstalk between different biosynthetic systems was predicted and experimentally proven. Furthermore, key residues important for maintaining (or preventing) NRPS interaction were identified. Point mutation of one of these key residues within the acceptor COM domain of TycC1 was sufficient to shift its selectivity from the cognate donor COM of TycB3 toward the noncognate donor COM domain of TycB1. Finally, an artificial NRPS complex was constructed, constituted of enzymes derived from three different biosynthetic systems. By virtue of domain fusions, the interactions between all enzymes were established by the same set of COM domains. Because of the abrogated selectivity, this universal communication system was able to simultaneously form two biosynthetic complexes that catalyzed the combinatorial synthesis of different peptide products.
Proceedings of the National Academy of Sciences of the United States of America, Jan 2, 2004
Nonribosomal peptide synthetases (NRPSs) catalyze the formation of structurally diverse and biolo... more Nonribosomal peptide synthetases (NRPSs) catalyze the formation of structurally diverse and biologically important peptides. Given their modular organization, NRPSs provide an enormous potential for biocombinatorial approaches to generate novel bioactive compounds. Crucial for the exploitation of this potential is a profound knowledge of the intermolecular communication between partner NRPSs. The overall goal of this study was to understand the basis of protein-protein communication that facilitates the selective interaction in these multienzyme complexes. On this account, we studied the relevance of short regions at the termini of the NRPSs tyrocidine (Tyc) synthetases TycA, TycB, and TycC, constituting the Tyc biosynthetic template. In vitro and in vivo investigations of C-terminal deletion mutants of the initiation module TycA provided evidence for the existence and impact of short communication-mediating (COM) domains. Their decisive role in protein-protein recognition was subse...
ChemBioChem, 2006
Assembly of bioactive natural compounds through the action of nonribosomal peptide synthetases (N... more Assembly of bioactive natural compounds through the action of nonribosomal peptide synthetases (NRPSs) relies on the specific interplay of modules and domains along these multiple mega-enzymes. As the C termini of several bacterial NRPSs often harbor epimerization (E) domains that generate D-amino acids, these seem to facilitate the ordered intermolecular enzymatic interaction and the directed transfer of intermediates. To elucidate this bifunctional role, E domains in recombinant bimodular proteins derived from the tyrocidine synthetase B were investigated. By utilizing sequent tryptic proteolysis and HPLC Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), we could directly interrogate and determine the formation of intermediates attached to the TycB(3)-PCP domain of wild-type TycB(2-3) and to the E domain exchange enzyme TycB(2-3)-ATCAT/E(tycA). In addition, the two proteins and a version of TycB(2-3) fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with TycB(1) to corroborate E domain impact on intermodular NRPS interaction. Significant functional differences between the C-terminal aminoacyl- and peptidyl-E domains were observed in terms of in trans interaction and misinitiation. E domains originating from elongation modules (peptidyl-E domains) seem to be optimized for regulation of the progression of peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas E domains of initiation modules (aminoacyl-E domains) impair upstream condensation and cause misinitiation. The selection of E domains is therefore decisive for successful application in biocombinatorial engineering of nonribosomal peptides.
The interaction between enzymes of a nonribosomal peptide synthetase (NRPS) complex relies on the... more The interaction between enzymes of a nonribosomal peptide synthetase (NRPS) complex relies on the interplay of compatible sets of donor and acceptor communication-mediating (COM) domains. Hence, these domains are essential for the formation of a defined biosynthetic template, thereby directing the synthesis of a specific peptide product. Without the selectivity provided by different sets of COM domains, NRPSs should form random biosynthetic templates, which would ultimately lead to combinatorial peptide synthesis. This study aimed to exploit this inherent combinatorial potential of COM domains. Based on sequence alignments between COM domains, the crosstalk between different biosynthetic systems was predicted and experimentally proven. Furthermore, key residues important for maintaining (or preventing) NRPS interaction were identified. Point mutation of one of these key residues within the acceptor COM domain of TycC1 was sufficient to shift its selectivity from the cognate donor COM of TycB3 toward the noncognate donor COM domain of TycB1. Finally, an artificial NRPS complex was constructed, constituted of enzymes derived from three different biosynthetic systems. By virtue of domain fusions, the interactions between all enzymes were established by the same set of COM domains. Because of the abrogated selectivity, this universal communication system was able to simultaneously form two biosynthetic complexes that catalyzed the combinatorial synthesis of different peptide products.