Published May 23, 2023 | Version v1
Journal article Open

AsiteDesign: a Semirational Algorithm for an Automated Enzyme Design

  • 1. Barcelona Supercomputing Center (BSC), Barcelona 08034, Spain
  • 2. Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, D-17487 Greifswald, Germany
  • 3. − Barcelona Supercomputing Center (BSC), Barcelona 08034, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, Spain
  • 4. Barcelona Supercomputing Center (BSC), Barcelona 08034, Spain; Biomatter Designs, Vilnius 09120, Lithuania


With advances in protein structure predictions, the number of available high-quality structures has increased dramatically. In light of these advances, structure-based enzyme engineering is expected to become increasingly important for optimizing biocatalysts for industrial processes. Here, we present AsiteDesign, a Monte Carlo-based protocol for structure-based engineering of active sites. AsiteDesign provides a framework for introducing new catalytic residues in a given binding pocket to either create a new catalytic activity or alter the existing one. AsiteDesign is implemented using pyRosetta and incorporates enhanced 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.


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