Phenylalanine ammonia-lyase (PAL) is a homotetrameric enzyme of prokaryotic origin with significant therapeutic and biochemical relevance, catalyzing the nonoxidative deamination of L-phenylalanine to trans-cinnamic acid and ammonia. While its active site architecture and substrate selectivity have been extensively characterized, the dynamic behavior of the unstructured loop regions surrounding the active site remains poorly understood. These loops, particularly the lid-like loop containing a critical catalytic tyrosine residue, are thought to regulate substrate ingress, product egress, and catalytic efficiency. In this study, we investigate the functional consequences of modulating the flexibility of these loops through disulfide bond engineering. To predict optimal disulfide bond placements, we employed four distinct strategies. Three in-house approaches leveraged molecular dynamics simulations: (i) quantifying pair interaction energies via electrostatic and van der Waals forces, (ii) generating a contact map of residues within 5Å proximity, and (iii) implementing a machine-learning model trained on datasets from PDBCYS, SPX, and an internal database to rank cysteine pair likelihood within disulfide bond geometric constraints. Additionally, we utilized the publicly available Disulfide by Design web server as a comparative approach. Our machine-learning-guided strategy yielded a successful variant with high interchain oxidation efficiency in E. coli cytoplasm. Experimental analyses revealed that reducing the flexibility of a lid-like loop over the active site alters the activity and overall stability of PAL. Kinetic and molecular dynamics analyses revealed that this modification alters enzymatic activity by limiting the conformational dynamics necessary for optimal substrate accommodation. Our findings underscore the delicate balance between enzyme flexibility and catalytic efficiency, providing novel insights into the role of dynamic loop regions in PAL function.
