ABSTRACT: With advances in protein structure predictions, the number of available high-quality structures has increaseddramatically. In light of these advances, structure-based enzyme engineering is expected to become increasingly important foroptimizing biocatalysts for industrial processes. Here, we present AsiteDesign, a Monte Carlo-based protocol for structure-basedengineering of active sites. AsiteDesign provides a framework for introducing new catalytic residues in a given binding pocket toeither create a new catalytic activity or alter the existing one. AsiteDesign is implemented using pyRosetta and incorporatesenhanced sampling techniques to efficiently explore the search space. The protocol was tested by designing an alternative catalytic triad in the active site of Pseudomonas fluorescens esterase (PFE). The designed variant was experimentally verified to be active demonstrating that AsiteDesign can find alternative catalytic triads. Additionally, the AsiteDesign protocol was employed to enhance the hydrolysis of a bulky chiral substrate (1-phenyl-2-pentyl acetate) by PFE. The experimental verification of the designed variants demonstrated that F158L/F198A and F125A/F158L mutations increased the hydrolysis of 1-phenyl-2-pentyl acetate from 8.9 to 66.7 and 23.4%, respectively, and reversed the enantioselectivity of the enzyme from (R) to (S)-enantiopreference, with 32 and 55% enantiomeric excess (ee), respectively.