Design and Evolution of New Catalytic Activity with an Existing Protein Scaffold
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.
Metal: ZnLigand type: Amino acidHost protein: Glyoxalase II (Human)Anchoring strategy: DativeOptimization: GeneticReaction: Hydrolytic cleavageMax TON: ---ee: ---PDB: 2F50Notes: kcat/KM ≈ 184 M-1*s-1