Figure 7: Screening process for identifying an engineered KIVD with altered substrate specificity.

The design process started with identifying active-site residues of KIVD (depicted in red). These amino acids were allowed to either remain native or sample any of 11 relatively hydrophobic amino acids. A total of 10,000 design simulations were run and the amino acids identified in the 50 lowest-energy designs were used to guide construction of a small library of roughly 400 KIVD mutants from the original combinatorial space of 10¹¹ possible active-site mutations. Overall, 1,200 clonal isolates were screened for activity and specificity. The KIVD_VLV mutant was selected and subsequently purified for in vitro kinetic constant characterization.