Huguenin-Dezot, N. et al. Nature 565, 112–117 (2019).

Many enzymatic reactions, including those of serine hydrolases and cysteine proteases, proceed via the formation of an unstable acyl-enzyme intermediate. Because these intermediates exist in fleeting states, they are hard to study structurally, which prevents mechanistic analysis. Huguenin-Dezot et al. describe a trapping strategy for stabilizing such intermediate enzyme states. By using genetic code expansion methods, they incorporate the unnatural amino acid DAP (2,3-diaminopropionic acid) into an enzyme of interest, in place of the catalytic serine or cysteine residue. DAP acts as a nucleophile, forming an amide bond with the enzyme substrate, and effectively trapping the acyl-enzyme intermediate. The trapped intermediate states are more amenable to structure determination, such as by X-ray crystallography. The authors applied this approach to study the mechanism by which valinomycin synthetase oligomerizes and cyclizes its tetradepsipetidyl substrate to produce the natural product valinomycin.