Kim, H.S.

Science 2006, 311, 535-538, 10.1126/science.1118953

The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce β-lactamase activity into the αβ/βα metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo β-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (kcat /Km)app of 1.8 × 102 (mole/liter)–1 second–1, thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.

Chen, Y.; Wang, C.

Angew. Chem. Int. Ed. 2020, 59, 3444-3449, 10.1002/anie.201912962

The diverse secondary structures of nucleic acids are emerging as attractive chiral scaffolds to construct artificial metalloenzymes (ArMs) for enantioselective catalysis. DNA‐based ArMs containing duplex and G‐quadruplex scaffolds have been widely investigated, yet RNA‐based ArMs are scarce. Here we report that a cyclic dinucleotide of c‐di‐AMP and Cu2+ ions assemble into an artificial metalloribozyme (c‐di‐AMP⋅Cu2+) that enables catalysis of enantioselective Friedel–Crafts reactions in aqueous media with high reactivity and excellent enantioselectivity of up to 97 % ee. The assembly of c‐di‐AMP⋅Cu2+ gives rise to a 20‐fold rate acceleration compared to Cu2+ ions. Based on various biophysical techniques and density function theory (DFT) calculations, a fine coordination structure of c‐di‐AMP⋅Cu2+ metalloribozyme is suggested in which two c‐di‐AMP form a dimer scaffold and the Cu2+ ion is located in the center of an adenine‐adenine plane through binding to two N7 nitrogen atoms and one phosphate oxygen atom.