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Total Biosynthesis of the Pyrrolo[4,2]benzodiazepine Scaffold Tomaymycin on an In Vitro Reconstituted NRPS System.
- Source :
-
Cell Chemical Biology . Oct2017, Vol. 24 Issue 10, p1216-1227.e8. 1p. - Publication Year :
- 2017
-
Abstract
- Summary In vitro reconstitution and biochemical analysis of natural product biosynthetic pathways remains a challenging endeavor, especially if megaenzymes of the nonribosomal peptide synthetase (NRPS) type are involved. In theory, all biosynthetic steps may be deciphered using mass spectrometry (MS)-based analyses of both the carrier protein-coupled intermediates and the free intermediates. We here report the “total biosynthesis” of the pyrrolo[4,2]benzodiazepine scaffold tomaymycin using an in vitro reconstituted NRPS system. Proteoforms were analyzed by liquid chromatography (LC)-MS to decipher every step of the biosynthesis on its respective megasynthetase with up to 170 kDa in size. To the best of our knowledge, this is the first report of a comprehensive analysis of virtually all chemical steps involved in the biosynthesis of nonribosomally synthesized natural products. The study includes experiments to determine substrate specificities of the corresponding A-domains in competition assays by analyzing the adenylation step as well as the transfer to the respective carrier protein domain. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 24519456
- Volume :
- 24
- Issue :
- 10
- Database :
- Academic Search Index
- Journal :
- Cell Chemical Biology
- Publication Type :
- Academic Journal
- Accession number :
- 125706146
- Full Text :
- https://doi.org/10.1016/j.chembiol.2017.08.001