Roelfes, G.

ChemBioChem 2020, 21, 3077-3081, 10.1002/cbic.202000306

We have examined the potential of the noncanonical amino acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug-resistance regulator (LmrR) by stop codon suppression methodology. LmrR_HQAla was shown to complex efficiently with three different metal ions, CuII, ZnII and RhIII to form unique artificial metalloenzymes. The catalytic potential of the CuII-bound LmrR_HQAla enzyme was shown through its ability to catalyse asymmetric Friedel-Craft alkylation and water addition, whereas the ZnII-coupled enzyme was shown to mimic natural Zn hydrolase activity.

Roelfes, G.

Dalton Trans. 2017, 46, 4325-4330, 10.1039/C7DT00533D

An artificial metalloenzyme containing both a regulatory and a catalytic domain is selectively activated in presence of Fe2+ ions.

Roelfes, G.

ChemCatChem 2020, 12, 3190-3194, 10.1002/cctc.202000245

The supramolecular approach is among the most convenient methodologies for creating artificial metalloenzymes (ArMs). Usually this approach involves the binding of a transition metal ion complex to a biomolecular scaffold via its ligand, which also modulates the catalytic properties of the metal ion. Herein, we report ArMs based on the proteins CgmR, RamR and QacR from the TetR family of multidrug resistance regulators (MDRs) and Cu2+ ions, assembled without the need of a ligand. These ArMs catalyze the enantioselective vinylogous Friedel-Crafts alkylation reaction with up to 75 % ee. Competition experiments with ethidium and rhodamine 6G confirm that the reactions occur in the chiral environment of the hydrophobic pocket. It is proposed that the Cu2+-substrate complex is bound via a combination of electrostatic and π-stacking interactions provided by the second coordination sphere. This approach constitutes a fast and straightforward way to assemble metalloenzymes and may facilitate future optimization of the protein scaffolds via mutagenesis or directed evolution approaches.

Harada, A.; Yamaguchi, H.

Sci. Rep. 2019, 9, 10.1038/s41598-019-49844-0

Design and engineering of protein scaffolds are crucial to create artificial metalloenzymes. Herein we report the first example of C-C bond formation catalyzed by artificial metalloenzymes, which consist of monoclonal antibodies (mAbs) and C2 symmetric metal catalysts. Prepared as a tailored protein scaffold for a binaphthyl derivative (BN), mAbs bind metal catalysts bearing a 1,1?-bi-isoquinoline (BIQ) ligand to yield artificial metalloenzymes. These artificial metalloenzymes catalyze the Friedel-Crafts alkylation reaction. In the presence of mAb R44E1, the reaction proceeds with 88% ee. The reaction catalyzed by Cu-catalyst incorporated into the binding site of mAb R44E1 is found to show excellent enantioselectivity with 99% ee. The protein environment also enables the use of BIQ-based catalysts as asymmetric catalysts for the first time.

Roelfes, G.

Angew. Chem. Int. Ed. 2021, 60, 5913-5920, 10.1002/anie.202014771

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.