期刊
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
卷 60, 期 11, 页码 5913-5920出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202014771
关键词
artificial metalloenzymes; biocatalysis; copper; in cell NMR spectroscopy; in vivo catalysis
资金
- Netherlands Organisation for Scientific Research (NWO) [700.26.121, 700.10.443, 724.013.003]
- Netherlands' Magnetic Resonance Research School (NMARRS) [022.005.029]
- Netherlands Ministry of Education, Culture and Science [024.001.035]
The study demonstrates the supramolecular assembly of ArMs in E. coli cells and their application in enzyme catalysis. Improved mutants were obtained through directed evolution, and the whole-cell ArM system showed catalytic activity without the need for engineering.
We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)-phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel-Crafts alkylation of indoles and the Diels-Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.
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