Researchers have identified the long-sought proton donor in enoyl-thioester reductase (ETR) enzymes, enabling them to reengineer the stereochemistry of the enzymes’ catalytic action. The work has implications for polyketide synthase-based synthesis of natural products and natural-product-like compounds because related reductases are major components of polyketide synthase enzyme complexes. In reactions catalyzed by ETRs, a proton from an active-site amino acid or water molecule is transferred to the α-carbon of the enoyl-thioester substrate. But scientists haven’t been able to pin down the identity of the proton donor. Tobias J. Erb of the Max Planck Institute for Terrestrial Microbiology, in Marburg, Germany, and coworkers provided experimental evidence for an earlier proposal that an intermediate called a C2-ene adduct forms between the substrate and NADPH in the reaction, suggesting it might be involved in the proton-transfer process. Erb and coworkers’ use of this C2-ene adduct as a molecular probe of the reaction has now enabled them to identify an active-site tyrosine as the elusive proton donor (Nat. Chem. Biol. 2015, DOI: 10.1038/nchembio.1794). This new understanding also enabled them to reengineer the enzyme to catalyze the reaction with inverted stereochemistry.