Extended Data Fig. 1: Strategies for Dap-mediated hydrolase substrate discovery.

Genetically encoded pc-Dap in place of the catalytic serine or cysteine in the active site of a hydrolase enables the deprotection of pc-Dap to Dap and the activation of a hydrolase substrate trap that can covalently capture substrates. Purified recombinant and pre-activated Dap-containing hydrolase can be added to lysate or extract and used to capture substrates. Hydrolase containing pc-Dap can also be expressed directly in mammalian cells. The hydrolase substrate trap can then be optically activated in live cells to capture substrates. Covalent conjugates can be enriched by immunoprecipitation with stringent washing to remove non-covalent binders and the conjugates can be visualized and identified by immunoblot and mass spectrometry-based methods. Control experiments use the wild-type (WT) enzyme that does not form a stable covalent acyl-enzyme conjugate and the catalytically inactive mutant (Ala, catalytic serine/cysteine is mutated to alanine) of the hydrolase that does not react with substrates. We note that not all the Dap-containing hydrolase will necessarily be found in conjugates; in general, we expect the fraction of Dap-containing hydrolase in conjugates to be a function of hydrolase abundance, substrate abundance, effects on the rate of acyl-intermediate formation resulting from replacing the catalytic nucleophile with Dap, and the stability of the acyl-intermediate.