Systematic and Quantitative Analysis of Protein−Protein Recognition between Nonribosomal Peptide Synthetases Investigated in the Tyrocidine Biosynthetic Template
| Autor: | Daniel B. Stein, Henning D. Mootz, Mohamed A. Marahiel, Uwe Linne |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Tyrocidine
chemistry.chemical_classification Substrate Specificities Base Sequence Protein protein Molecular Conformation Biology Biochemistry Recombinant Proteins Anti-Bacterial Agents Kinetics chemistry.chemical_compound Enzyme chemistry Nonribosomal peptide Trans-acting Peptide Synthases Phosphorylation Gene Quantitative analysis (chemistry) DNA Primers |
| Zdroj: | Biochemistry. 42:5114-5124 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi034223o |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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