Activity Screening of Carrier Domains within Nonribosomal Peptide Synthetases Using Complex Substrate Mixtures and Large Molecule Mass Spectrometry
| العنوان: | Activity Screening of Carrier Domains within Nonribosomal Peptide Synthetases Using Complex Substrate Mixtures and Large Molecule Mass Spectrometry |
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| المؤلفون: | Paul D. Straight, Pieter C. Dorrestein, Daniel J. Edwards, William H. Gerwick, Neil L. Kelleher, Michael A. Fischbach, Christopher T. Walsh, Shaun McLaughlin, Myat T. Lin, Sylvie Garneau-Tsodikova, Jonathan R. Blackhall, Roberto Kolter |
| المصدر: | Biochemistry. 45:1537-1546 |
| بيانات النشر: | American Chemical Society (ACS), 2006. |
| سنة النشر: | 2006 |
| مصطلحات موضوعية: | Aminocoumarins, Biology, Mass spectrometry, Biochemistry, Yersiniabactin, Mass Spectrometry, Article, Fourier transform ion cyclotron resonance, Enterobactin, Substrate Specificity, chemistry.chemical_compound, Adenosine Triphosphate, Phenols, Nonribosomal peptide, Catalytic Domain, Sulfhydryl Compounds, Peptide Synthases, chemistry.chemical_classification, Clorobiocin, Substrate (chemistry), Bromine, Combinatorial chemistry, Pyrrolidinones, Coumermycin A1, Thiazoles, Aminoglycosides, chemistry, Multigene Family, Novobiocin, Bacillus subtilis |
| الوصف: | For screening a pool of potential substrates that load carrier domains found in non-ribosomal peptide synthetases, large molecule mass spectrometry is shown to be an ideal, unbiased assay. Combining the high resolving power of Fourier-Transform Mass Spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin and enterobactin biosynthetic pathways as proof-of-principle, preferred substrates are readily identified from substrate pools. Furthermore this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the genes pksN and pksJ, that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate. |
| تدمد: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi052333k |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::6dd003c74c7df69fad569647a2d2c4cf https://doi.org/10.1021/bi052333k |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....6dd003c74c7df69fad569647a2d2c4cf |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15204995 00062960 |
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| DOI: | 10.1021/bi052333k |