Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid

Abstract

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 insight7–10, 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.

2,3-Diaminopropanols Obtained from d-Serine as Intermediates in the Synthesis of Protected 2,3-l-Diaminopropanoic Acid (l-Dap) Methyl Esters

“…l-Dap dipeptides were used as reference structures to investigate the ionizability of side-chain primary amino functions in amino acids [24], and the substitution of aspartic units by l-Dap was adopted in the synthetic preparation of glycoproteins [25]. l-Dap acts as crosslinker in graphene sponges of clinical importance for hemostasis control [26], and it is a component of biosynthetic model compounds employed in studies aiming to define acyl-enzyme activities and foldings [27,28]. l-Dap-based peptide dendrimers catalyze asymmetric processes in organic synthesis [29,30].…”

Enzymes engineered to trap reaction intermediates

“…Flu continues to pose a threat to human health, and there is a pressing need to develop countermeasures that can confer broad protection against the different flu strains. Moreover, vaccines are usually less effective at providing protection for children and for older individuals than they are for the rest of the population 2 .…”

“…In this approach, enzymes called amino acyl-tRNA synthetases are designed to load noncanonical amino acids (ncAAs) onto transfer RNAs, so that the ncAAs can be incorporated into proteins by the cell's ribosome apparatus 1 . On page 112, Huguenin-Dezot et al 2 report a method for studying enzyme mechanisms that uses genetic-code expansion in a new way. The authors demonstrate the utility of this approach using an enzyme that produces an antibiotic.…”

“…This produces an acyl-enzyme intermediate, which is subsequently hydrolysed to release the depsipeptide. b, Huguenin-Dezot et al 2 report a method in which the nucleophilic residue is substituted by the 2,3-diaminopropionic acid (DAP) residue -which here replaces the hydroxyl (OH) group of serine with an amine (NH 2 ) group. The resulting acyl-enzyme intermediate resists hydrolysis, allowing it to be visualized using X-ray crystallography.…”

A Light‐Activated Acyl Carrier Protein “Trap” for Intermediate Capture in Type II Iterative Polyketide Biocatalysis

“…[14] Further work will be required to corroborate the preliminary insights gathered by our experiments. Nonetheless, the ACP probe 8 herein prepared and evaluated constitutes ar are example of protein "trap" for biosynthetic species [31] and the first for polyketide synthases. Its use in conjunction with advanced FT-ICR-MS analyses and data analysis tools represents an ew promising approachf or the study of challenging biosynthetic enzymes that make use of dynamic carrier proteins,i ncluding fatty acid synthases and nonribosomal peptidesynthetases.…”