Many enzymes catalyse reactions that proceed through covalent acyl-enzyme (ester or thioester) intermediates1. These enzymes include serine hydrolases2,3 (encoded by one per cent of human genes, and including serine proteases and thioesterases), cysteine proteases (including caspases), and many components of the ubiquitination machinery4,5. Their important acyl-enzyme intermediates are unstable, commonly having half-lives of minutes to hours6. In some cases, acyl-enzyme complexes can be stabilized using substrate analogues or active-site mutations but, although these approaches can provide valuable insight7,8,9,10, they often result in complexes that are substantially non-native. Here we develop a strategy for incorporating 2,3-diaminopropionic acid (DAP) into recombinant proteins, via expansion of the genetic code11. We show that replacing catalytic cysteine or serine residues of enzymes with DAP permits their first-step reaction with native substrates, allowing the efficient capture of acyl-enzyme complexes that are linked through a stable amide bond. For one of these enzymes, the thioesterase domain of valinomycin synthetase12, we elucidate the biosynthetic pathway by which it progressively oligomerizes tetradepsipeptidyl substrates to a dodecadepsipeptidyl intermediate, which it then cyclizes to produce valinomycin. By trapping the first and last acyl-thioesterase intermediates in the catalytic cycle as DAP conjugates, we provide structural insight into how conformational changes in thioesterase domains of such nonribosomal peptide synthetases control the oligomerization and cyclization of linear substrates. The encoding of DAP will facilitate the characterization of diverse acyl-enzyme complexes, and may be extended to capturing the native substrates of transiently acylated proteins of unknown function.
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Source data for all figures are available from the corresponding authors upon reasonable request. The models and structure factors for the crystal structures are deposited in the Protein Data Bank with accession numbers 6ECB, 6ECC, 6ECD, 6ECE and 6ECF. Detailed methods, including chemical syntheses, are available in the Supplementary Information.
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We thank P. Emsley and G. Murshudov for their help in modelling depsipeptides; A. Wahba, J. Reimer, G. Bridon, K. Heesom and T. Elliott for help with mass spectrometry; M. Tarry and N. Rogerson for editing; C. Alonso for early crystal trials; R. Hay for early discussions on DAP; and S. Zhang for help with cloning. We also thank the staff of beamlines CLS 08ID-1 (S. Labiuk, J. Gorin, M. Fodje, K. Janzen, D. Spasyuk and P. Grochulski) and APS 24-ID-C (grants GM124165, RR029205, DE-AC02-06CH11357; F. Murphy). This work is supported by grants to J.W.C. from the Medical Research Council, UK (grants MC_U105181009 and MC_UP_A024_1008), to T.M.S. from the Canada Research Chair and the Natural Sciences and Engineering Research Council of Canada (NSERC; Discovery Grant 418420) and to C.N.B. from the NSERC (Discovery Grant 06167).
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