Leveraging intrinsic flexibility to engineer enhanced enzyme catalytic activity

Abstract

Significance Protein flexibility has been recognized as a key contributor to enzyme evolution and catalytic activity. Several studies have illustrated how amino acid substitutions that affect protein flexibility can impact catalysis. However, it is unknown whether structural information regarding conformational flexibility can be exploited for directed evolution of enzymes with higher catalytic activity. Using as a model human kynureninase, an enzyme with important therapeutic implications in cancer immunotherapy, we show that mutagenesis of residues exclusively located within high B-factor regions distal to the active site resulted in a variant with markedly enhanced catalytic activity for its nonpreferred substrate, kynurenine. Our results suggest that modulation of intrinsic flexibility through mutagenesis of remote flexible regions constitutes a promising strategy for directed enzyme evolution.