GRACE: Generative Redesign in Artificial Computational Enzymology

Designing <i>de novo</i> enzymes is complex and challenging, especially to maintain the activity. This research focused on motif design to identify the crucial domain in the enzyme and uncovered the protein structure by molecular docking. Therefore, we developed a Generative Redesign in Artificial Computational Enzymology (GRACE), which is an automated workflow for reformation and creation of the <i>de novo</i> enzymes for the first time. GRACE integrated RFdiffusion for structure generation, ProteinMPNN for sequence interpretation, CLEAN for enzyme classification, and followed by solubility analysis and molecular dynamic simulation. As a result, we selected two gene sequences associated with carbonic anhydrase from among 10,000 protein candidates. Experimental validation confirmed that these two novel enzymes, <i>i.e.</i>, dCA12_2 and dCA23_1, exhibited favorable solubility, promising substrate-active site interactions, and achieved activity of 400 WAU/mL. This workflow has the potential to greatly streamline experimental efforts in enzyme engineering and unlock new avenues for rational protein design.