Liang_2025_ACS.Omega_10_21693

Pregabalin has garnered extensive clinical application for the management of neuropathic pain and epilepsy owing to its high efficacy and broad drug concentration range. EstZF172 is a key enzyme in the biosynthesis of pregabalin, capable of stereoselectively catalyzing the production of (R)-CCMA from the key intermediate rac-CNDE. The novel crystal structure of EstZF172 indicates that it contains a highly conserved Ser-Lys-Tyr catalytic triad and belongs to the family VIII(2) carboxylesterases. Molecular docking demonstrates that the steric hindrance presented by residues I159 and F239 plays a crucial role in influencing the binding affinity of the chiral substrate (R)-CNDE for the catalytic site. The study provides a structural basis and reference for the stereoselective catalysis of EstZF172 and engineering modification of the key enzyme in the synthesis of pregabalin.