Identifying a cooperative catalytic network for efficient esterase catalysis

Abstract

Active-site redesign frequently yields modest improvements because residues controlling physical steps like substrate binding and product release lie outside the active site. Efficient catalysis requires a cooperative catalytic network of residues that support both the chemical and physical steps of catalysis. Using ancestral hydroxynitrile lyase HNL1, an /{beta}-hydrolase with poor esterase activity, we tested this framework directly. Matching all active-site residues to a proficient esterase improved KM five-fold but left kcat unchanged, confirming that chemical machinery alone is insufficient. Activity-weighted sequence comparison (SigniSite) across ten homologous HNLs and esterases identified 38 positions disfavoring esterase activity. Experimental refinement yielded a minimal set of fifteen substitutions (HNL1-15) with [~]60-fold higher kcat and [~]400-fold higher kcat/KM. Single-substitution reversion analysis confirmed that all fifteen substitutions are essential and provided evidence for strong cooperativity between them. X-ray crystal structures of HNL1 and HNL1-15 reveal three coordinated structural changes: reshaping the substrate-binding pocket to favor productive ester binding, restoring access to the oxyanion hole, and opening an additional tunnel for product egress and water entry. These changes arise through backbone rearrangements and altered flexibility rather than direct active-site contacts, explaining why the responsible positions escape conservation-based detection. Because cooperativity masks individual contributions, engineering such networks may require step-specific assays -- measuring binding, acylation, or product release directly -- rather than screening composite kcat.