Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747, AG, Groningen, The Netherlands.
NMR Spectroscopy group, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584, CH, Utrecht, The Netherlands.
Angew Chem Int Ed Engl. 2021 Mar 8;60(11):5913-5920. doi: 10.1002/anie.202014771. Epub 2021 Feb 1.
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.
我们报告了基于乳球菌多药耐药调节剂（LmrR）和外源铜（II）-菲咯啉配合物的人工金属酶（ArM）在大肠杆菌细胞细胞质中的超分子组装。通过催化、细胞分级分离和抑制剂实验的组合，辅以细胞内固态 NMR 光谱学，证实了细胞内的组装。含有 ArM 的完整细胞在催化吲哚的对映选择性 Frie-del-Crafts 烷基化和氮杂查尔酮与环戊二烯的 Diels-Alder 反应中具有活性。定向进化导致两种不同的反应改进突变体，分别为 LmrR_A92E_M8D 和 LmrR_A92E_V15A。整个细胞 ArM 系统不需要对微生物宿主、蛋白质支架或辅因子进行工程改造即可实现 ArM 组装和催化。我们认为这是将非生物催化与生物合成相结合以产生混合代谢的关键一步。
