In conclusion, we have pursued the first in-depth biocatalytic and mechanistic study of a G3PS, thereby expanding the biocatalytic alkylation toolbox by the ultrastable ether synthase AfG3PS. Using high-throughput kinetics and biophysical methods, we profiled AfG3PS and selected mutants, outlining principles governing its substrate scope and preferences. Using synthetic chemistry and spectroscopy, we further demonstrated that this enzyme makes various (E)-and (Z)-configured prenyl glycerol ethers while exerting perfect control over the configuration at the glycerol unit. Our mechanistic data highlight the sensitive nature of its active site and provide multifacted data facilitating its future engineering. Ultimately, we believe that (engineered) ether synthases such as AfG3PS will prove powerful catalysts, unfolding their full potential in heterogenous processes and enzymatic cascades.