An engineered disulfide staple restricts lid loop dynamics and alters substrate specificity of phenylalanine ammonia-lyase
In Anabaena variabilis (Trichormus variabilis) phenylalanine ammonia-lyase (AvPAL), a conserved lid-like loop sits over the active site and has been studied both for its role in positioning a catalytic tyrosine and for its contribution to phenylalanine aminomutase (PAM) activity. While the active site architecture and substrate specificity of AvPAL have been extensively characterized, the dynamic behavior of this unstructured loop beyond its role in catalysis remains poorly understood. Here, we investigate the functional role of this loop by restricting its mobility through targeted interchain disulfide bond engineering. Three in-house approaches were designed to predict ideal cysteine residue pairs: (i) quantifying pair interaction energies via electrostatic and van der Waals forces, (ii) generating a contact map of residues within 5 [A] 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. Our machine-learning-guided strategy yielded a successful variant with complete oxidation efficiency in E. coli. Rigidification of this loop reveals that it also functions as a regulator of substrate specificity. Multi-scale molecular simulation analyses (molecular dynamics, metadynamics, quantum/molecular mechanics) reveal that this modification alters the active-site pocket by reducing the conformational dynamics of substrate binding. Our findings underscore the delicate balance between enzyme flexibility and catalytic efficiency, providing novel insights into the role of this understudied dynamic loop region in AvPAL.