Scientists design covalent-intermediate enzyme

Researchers reported in *Science* on February 13, 2025 that they designed enzymes from scratch that carry out a two-step reaction through a covalent intermediate, a mechanism widely used by natural enzymes such as proteases. The work centered on serine hydrolases, a large enzyme family that breaks chemical bonds through a catalytic serine residue. The authors said the advance came from designing the protein around the active site, rather than trying to fit a designed active site into an existing natural scaffold. The paper was led by Anna Lauko and included researchers tied to the University of Washington, Howard Hughes Medical Institute, UC Santa Barbara, UCSF and the University of Pittsburgh. If you’re trying to understand why this drew attention, the key claim is not just “new enzyme design.” The paper says the team designed catalysts that can accommodate multiple states along a reaction path, including two tetrahedral intermediates and an acyl-enzyme intermediate, instead of optimizing only for a single transition state. That matters because many natural enzymes do chemistry in steps, with short-lived intermediates formed and then consumed before the final product appears. (eurekalert.org) ### What is a covalent intermediate, in plain terms? A covalent intermediate is a temporary chemical state in which the enzyme briefly forms a bond with the molecule it is transforming. In serine hydrolases, the paper says, a serine residue in the active site attacks the substrate and forms an acyl-enzyme intermediate before water helps release the final product. The AAAS press summary described the mechanism as analogous to what many proteases do when they break apart proteins. (science.org) That is different from a simpler catalytic design in which the enzyme only stabilizes one fleeting transition state. The authors argued that designing for the whole catalytic cycle is necessary for reactions that proceed through several distinct geometries. ### How did the researchers say they built these enzymes? The Science paper said the team used RFdiffusion to generate protein structures tailored to a desired active site, then used a neural-network method called PLACER to test whether those designs stayed compatible with each step of the catalytic cycle. (eurekalert.org) The AAAS press package said PLACER predicts atomic structures of enzyme active sites by considering the protein backbone, amino-acid identities and bound molecules. (science.org) UC Santa Barbara described the broader workflow as starting with a helical-bundle framework, designing amino-acid sequences for the target structure, checking structures with x-ray crystallography and then refining the designs further. Yang Yang, a UC Santa Barbara chemistry professor and senior author, said the collaboration produced “very efficient and very selective enzymes” from a simple miniature helical-bundle protein in a proof-of-concept study. (eurekalert.org) ### What did they actually make? The model reaction was ester hydrolysis, which the authors chose because serine hydrolases use a complex active site and multiple reaction states that make the system a demanding test case. The paper reported functional designed serine hydrolases and said the active designs spanned five distinct folds unlike those found in natural serine hydrolases. (news.ucsb.edu) The authors said that result expands the known fold space for this enzyme class. In the paper’s wording, the approach provides “a roadmap” for designing enzymes that catalyze multistep transformations. ### Why is this different from older enzyme-design efforts? Previous enzyme-design efforts often started with pre-existing protein backbones and tried to place catalytic residues into them. (eurekalert.org) The Science paper said that strategy can limit geometric precision and active-site preorganization, which in turn has held down catalytic activity in many designed enzymes. By contrast, this study built proteins to suit the active site and then screened them against several catalytic states, not just one. (science.org) The AAAS press summary said that framework could help engineers build enzymes for more complex, multistep reactions. ### What comes next? The paper is already published in *Science* under the title “Computational design of serine hydrolases,” DOI 10.1126/science.adu2454. (science.org) UC Santa Barbara said the same workflow could be used to combine desirable properties into “new-to-nature” catalysts for applications including drug development and materials design, though those applications were described as future use rather than results demonstrated in this study. (news.ucsb.edu) (eurekalert.org)