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.

Nonribosomal peptide synthetases (NRPSs) synthesize a vast variety of small molecules, including antibiotics, antitumors, and immunosuppressants. The NRPS condensation (C) domain catalyzes amide bond formation, the central chemical step in nonribosomal peptide synthesis. The catalytic mechanism and substrate determinants of the reaction are under debate. We developed chemical probes to structurally study the NRPS condensation reaction. These substrate analogs become covalently tethered to a cysteine introduced near the active site, to mimic covalent substrate delivery by carrier domains. They are competent substrates in the condensation reaction and behave similarly to native substrates. Co-crystal structures show C domain-substrate interactions, and suggest that the catalytic histidine's principle role is to position the α-amino group for nucleophilic attack. Structural insight provided by these co-complexes also allowed us to alter the substrate specificity profile of the reaction with a single point mutation.

This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature's AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections.Nonribosomal depsipeptides are natural products composed of amino and hydroxy acid residues. The hydroxy acid residues often derive from α-keto acids, reduced by ketoreductase domains in the depsipeptide synthetases. Biochemistry and structures reveal the mechanism of discrimination for α-keto acids and a remarkable architecture: flanking intact adenylation and ketoreductase domains are sequences separated by >1100 residues that form a split "pseudoAsub" domain, structurally important for the depsipeptide module's synthetic cycle.Nonribosomal peptide synthetases (NRPSs) are multi-domain enzymes that produce a vast array of biologically-active compounds, including clinically-used therapeutics such as the antibiotic daptomycin, the immunosuppressant cyclosporin and the antifungal caspofungin 1 . NRPSs are composed of a series of modules, sets of domains that work together to add a building block substrate to the growing peptide chain, in a manner analogous to assembly lines.Depsipeptides contain both amino acid residues and hydroxy acid residues, linked by amide and ester bonds, respectively. Important depsipeptides include the piscicide antimycin, the K+ ionophores cereulide 2,3 and valinomycin 4,5 (Supplementary Fig. 1), the anticancer agent cryptophycin 6 , the antimicrobial kutzneride 7 , the antifungal hectochlorin 8 and the insecticide mycotoxins bassianolide and beauvericin 9 . The hydroxy acid residues in fungal compounds bassianolide and beauvericin originate from direct selection and incorporation of α-hydroxy acids, but the hydroxy acid residues in the bacterial compounds antimycin, hectochlorin, kutzneride, cryptophycin, cereulide and valinomycin are derived from α-keto acid substrates 10 .In bacterial depsipeptide synthetases, modules responsible for adding hydroxy acids include condensation (C) (if an elongation module), adenylation (A), ketoreductase (KR), and peptidyl carrier protein (PCP) domains (Fig. 1a and Supplementary Fig. 1) [11][12][13][14] . These A domains select α-keto acids using a hitherto unknown mechanism to differentiate them from α-amino and α-hydroxy acids.Intriguingly, the aspartate which contacts the α-amino group in amino acid-selecting A domains is altered to a hydrophobic residue in α-keto acid-selecting A domains, not to a positive or polar residue 7,10,11 . Depsipeptide synthetase A domains adenylate the α-keto acid, then transfer it to the PCP domain. The PCP domain transports the α-keto acyl moiety to the KR domain for stereoselective reduction. After reduction, the α-hydroxyl acyl-PCP moves to that module's C domain for condensation, making an ester bond (or goes directly to the downstream C domain in the case of A-KR-PCP initiation modules), and synthesis continues as in the canonical case.Ketoreducing depsipeptide modul...

Depsipeptides are compounds that contain both ester bonds and amide bonds. Important natural product depsipeptides include the piscicide antimycin, the K + ionophores cereulide and valinomycin, the anticancer agent cryptophycin, and the antimicrobial kutzneride. Furthermore, database searches return hundreds of uncharacterized systems likely to produce novel depsipeptides. These compounds are made by specialized nonribosomal peptide synthetases (NRPSs). NRPSs are biosynthetic megaenzymes that use a module architecture and multi-step catalytic cycle to assemble monomer substrates into peptides, or in the case of specialized depsipeptide synthetases, depsipeptides. Two NRPS domains, the condensation domain and the thioesterase domain, catalyze ester bond formation, and ester bonds are introduced into depsipeptides in several different ways. The two most common occur during cyclization, in a reaction between a hydroxy-containing side chain and the C-terminal amino acid residue in a peptide intermediate, and during incorporation into the growing peptide chain of an α-hydroxy acyl moiety, recruited either by direct selection of an α-hydroxy acid substrate or by selection of an α-keto acid substrate that is reduced in situ. In this article, we discuss how and when these esters are introduced during depsipeptide synthesis, survey notable depsipeptide synthetases, and review insight into bacterial depsipeptide synthetases recently gained from structural studies.

Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride.