MechFind: A computational framework for de novo prediction of enzyme mechanisms.

Despite the importance of understanding the step-by-step mechanism of enzymatically catalyzed reactions, fewer than one thousand cataloged mechanistic annotations can be found in the open literature and databases. Herein, we introduce MechFind, a computational tool that generates elementally and charge-balanced putative enzyme mechanisms. Unlike previous methods that require structural data or user-supplied active site residues, MechFind uses only the overall reaction stoichiometry as input, abstracting individual reaction steps as the gain or loss of chemical moieties. An optimization framework then identifies the top ten most parsimonious (i.e., fewest steps) mechanistic descriptions for the overall transformation, which are then re-ranked based on their mechanistic similarity to a database of known, curated mechanisms. MechFind recovered the correct mechanism for 72% of the Mechanism and Catalytic Site Atlas (M-CSA) training dataset within the top ten predictions and was independently validated on six enzyme mechanisms absent from the training set. When deployed at scale on 28,412 reactions from the Rhea database, MechFind identified a plausible mechanism for 64% of all entries, generating over 18,000 novel mechanistic hypotheses expanding significantly upon the current state of the art in prediction (i.e., a 20-fold increase). By proposing detailed reaction mechanisms MechFind provides information that can be leveraged by modern ML-based de novo protein design tools, offering a resource for improving functional annotation and accelerating the engineering of novel biocatalysts. All codes, curated datasets, and results are available at https://github.com/maranasgroup/MechFind.