期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 143, 期 7, 页码 2736-2740出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.1c00925
关键词
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资金
- ETH Zurich
- Swiss National Research Foundation
This study demonstrates a minimally invasive strategy for efficient functionalization of NRPs on the pathway level through selective incorporation of different functional groups by single point mutations. The approach allows site-selective fluorescent labeling of NRPs and holds great potential for NRP isolation, molecular target elucidation, and combinatorial optimization of NRP therapeutics by combining synthetic biology with bioorthogonal chemistry.
Nonribosomal peptides (NRPs) are a therapeutically important class of secondary metabolites that are produced by modular synthetases in assembly-line fashion. We previously showed that a single Trp-to-Ser mutation in the initial Phe-loading adenylation domain of tyrocidine synthetase completely switches the specificity toward clickable analogues. Here we report that this minimally invasive strategy enables efficient functionalization of the bioactive NRP on the pathway level. In a reconstituted tyrocidine synthetase, the W227S point mutation permitted selective incorporation of Phe analogues with alkyne, halogen, and benzoyl substituents by the initiation module. The respective W2742S mutation in module 4 similarly permits efficient incorporation of these functionalized substrate analogues at position 4, expanding this strategy to elongation modules. Efficient incorporation of an alkyne handle at position 1 or 4 of tyrocidine A allowed site-selective one-step fluorescent labeling of the corresponding tyrocidine analogues by Cu(I)-catalyzed alkyne-azide cycloaddition. By combining synthetic biology with bioorthogonal chemistry, this approach holds great potential for NRP isolation and molecular target elucidation as well as combinatorial optimization of NRP therapeutics.
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