What it is
De novo enzyme design has advanced, but its successes have been largely limited to model reactions with low energy barriers. Amide bonds, such as those linking amino acids along a protein's backbone, are stable for hundreds of years in neutral water because hydrolysing them takes so much energy. The authors describe the de novo design of enzymes that use an activated cysteine nucleophile to cut the polypeptide backbone in a sequence-dependent manner, with a success rate of 13 of 69 designs, 19% and rate enhancements over the background reaction of up to 30 million-fold. The designed proteases have folds very different from those of natural proteases (TM score below 0.50), and six crystal structures are very close to the design models (Cα root mean squared deviation below 1.2 Å), which the authors read as showing the accuracy of the design method and its capacity to generalize. Experimental and computational analyses suggest that the remaining gap in activity to the most active natural cysteine proteases comes from imperfections in how the active site is pre-organized and how the substrate is positioned. The designed proteases efficiently cleave their targets in mammalian cells, which the authors say opens the door to a wide range of synthetic biology applications.
Why it matters
Designed from scratch, 13 of 69 cysteine proteases cut protein backbones in a sequence-dependent way, up to 30 million-fold faster than the background reaction. They also cleaved their targets in mammalian cells. Amide bonds are stable for hundreds of years in neutral water, and de novo enzymes had mostly catalysed easier model reactions. These proteases attack with an activated cysteine rather than the zinc-activated water of the designed metalloproteases Nature published the same day from an overlapping University of Washington team, their folds look little like any natural protease, and six crystal structures match the designs closely.
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Filed underProtein Structure and Dynamics, Enzyme Catalysis and Immobilization, Enzyme Structure and Function
