title: Systematic and quantitative analysis of protein-protein recognition between nonribosomal peptide synthetases investigated in the tyrocidine biosynthetic template.
abstract: We present a systematic and quantitative study of the protein-protein recognition between the three tyrocidine synthetases TycA, TycB, and TycC investigated with two artificial in trans assay systems, which had been previously developed: the "DKP assay system" for the interaction of TycA with TycB and the "L/D-Phe-L-Asn assay system" for the interaction of TycB with TycC. TycA-A(Phe)TE and TycB(3)-A(Phe)TE, which are used as donor enzymes, both provide D-Phe-S-Ppant, so that no substrate specificities interfered with the quantification of protein-protein recognition. We tested all donor/acceptor enzyme combinations between the two artificial assay systems for product formation activities as well as two hybrid enzymes, where the E-domains of TycA and TycB(3) had been exchanged against each other. Furthermore, four donor/acceptor protein fusions were constructed on gene level, resulting in dimodular proteins. We were able to show that the E-domains mediate protein-protein recognition in trans. Product formation of the different donor assayed with the two acceptor enzymes TycB(1)-CA(Pro)T and TycC(1)-CA(Asn)T/Te in trans was only obtained if the donor enzyme harbored the cognate E-domain. Interestingly, all in cis fusions (dimodular proteins) were active, giving strong evidence that unnatural protein-protein interactions can be "forced" by fusion of the distinct enzymes. Finally, we were able to detect product formation in the "DKP system" with engineered hybrid proteins where the A-domain of TycA had been exchanged against the isoleucine-activating A-domain of BacA(1) and the valine-activating A-domain of TycC(4), respectively. All of these findings are of high relevance for future NRPS engineering approaches.
authors.affiliation: Fachbereich Chemie/Biochemie, Philipps Universität Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Linne
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Stein
authors.forename: Henning D
authors.initials: HD
authors.lastname: Mootz
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry; D001483:Base Sequence; D017931:DNA Primers; D007700:Kinetics; D008968:Molecular Conformation; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D014440:Tyrocidine / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D011994:Recombinant Proteins; D014440:Tyrocidine; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase