Structural and mutational analysis of the nonribosomal peptide synthetase heterocyclization domain provides insight into catalysis Kristjan Bloudoffa, Christopher D. Fageb, Mohamed A. Marahielb, and T. Martin Schmeinga,1 aDepartment of Biochemistry, McGill University, Montreal, QC H3G 0B1, Canada; and bDepartment of Chemistry/Biochemistry, LOEWE Center for Synthetic Microbiology, Philipps-Universität Marburg, 35032 Marburg, Germany Edited by Peter B. Moore, Yale University, New Haven, CT, and approved November 30, 2016 (received for review August 31, 2016) Nonribosomal peptide synthetases (NRPSs) are a family of multido- rings are found in many peptides with important clinical and re- main, multimodule enzymes that synthesize structurally and func- search utility, such as the antibiotics bacitracin A (6) and zelko- tionally diverse peptides, many of which are of great therapeutic or vamycin (13), the antitumor agents bleomycin (14) and epothilone commercial value. The central chemical step of peptide synthesis is (8), the immunosuppressant argyrin (15), the siderophores myco- amide bond formation, which is typically catalyzed by the conden- bactin (16) and yersiniabactin (17), and the microbiome genotoxin sation (C) domain. In many NRPS modules, the C domain is replaced colibactin (18). Introduction of the five-membered heterocyclic by the heterocyclization (Cy) domain, a homologous domain that ring makes the peptide resistant to proteolytic cleavage and can performs two consecutive reactions by using hitherto unknown induce conformations in the peptide that favor interaction with catalytic mechanisms. It first catalyzes amide bond formation, and biological targets (19). then the intramolecular cyclodehydration between a Cys, Ser, or Thr In NRPSs that synthesize these heterocycle-containing products, side chain and the backbone carbonyl carbon to form a thiazoline, the module specific for the Cys, Ser, or Thr monomer substrate oxazoline, or methyloxazoline ring. The rings are important for the contains a Cy domain in place of the C domain. Cy domains are form and function of the peptide product. We present the crystal evolutionarily and structurally related to C domains (20). The Cy structure of an NRPS Cy domain, Cy2 of bacillamide synthetase, at a domain first catalyzes nucleophilic attack on the thioester of a PCP- BIOCHEMISTRY resolution of 2.3 Å. Despite sharing the same fold, the active sites linked donor substrate by the α-amino group of a Cys-, Ser-, or Thr- of C and Cy domains have important differences. The structure PCP substrate, presumably in a manner similar to C domains (6, 7, allowed us to probe the roles of active-site residues by using muta- 10–12, 21–23) (Fig. 1). In the two-step cyclodehydration reaction tional analyses in a peptide synthesis assay with intact bacillamide that follows, the thiol of the Cys side chain or hydroxyl of the Ser or synthetase. The drastically different effects of these mutants, inter- Thr side chain first attacks the carbonyl carbon of the newly formed preted by using our structural and bioinformatic results, provide amide to create the heterocycle (10, 11), and then the former car- insight into the catalytic mechanisms of the Cy domain and impli- bonyl oxygen is removed in a dehydration reaction, which intro- cate a previously unexamined Asp-Thr dyad in catalysis of the duces the carbon-nitrogen double bond of the thiazoline or (methyl) cyclodehydration reaction. oxazoline ring. The nascent heterocyclic peptidyl-PCP can be used as the donor substrate by the next module’s C domain, or is nonribosomal peptide synthetase | heterocyclization domain | X-ray first oxidized or reduced by discrete oxidase or reductase domains crystallography | bacillamide | mutational analysis (10, 24). onribosomal peptide synthetases (NRPSs) are a family of Significance Nlarge, multimodular enzymes that produce a range of im- portant bioactive secondary metabolites (1). NRPS products have Nonribosomal peptide synthetases produce peptides with wide great diversity because they can use more than 500 different acyl varieties of therapeutic and biological activities. Monomer sub- monomer substrates, including L-amino acids, D-amino acids, aryl strates are typically linked by a condensation domain. However, acids, fatty acids, hydroxy acids, and keto acids, and they can in many modules, a heterocyclization (Cy) domain takes its place subsequently modify these moieties during peptide synthesis. and performs both condensation and cyclodehydration of a cys- NRPSs function in a modular, assembly-line fashion. A typical teine, serine, or threonine to form a five-membered ring in the elongation module consists of a condensation (C), an adenylation peptide backbone. Although studied for decades, the mecha- (A), and a peptidyl-carrier protein (PCP) domain. The A domain nisms of condensation and cyclodehydration by Cy domains were specifically recognizes and activates a monomer acyl substrate previously unknown. The crystal structure of a Cy domain, and through adenylation, then transfers it onto the prosthetic phos- accompanying mutagenic and bioinformatics analyses, uncover phopantetheine arm of the PCP domain. This acyl-PCP then travels the importance of an aspartate and a threonine for the cyclo- to the C domain acceptor site for condensation with the upstream dehydration reaction. This study provides insight into the catal- module’s acyl-PCP at the C domain donor site (2–5). The PCP ysis of condensation by the Cy domain and enables the proposal domain brings the elongated peptide chain to the downstream of a reaction mechanism for cyclodehydration. module, where it is passed off and further elongated in the next condensation reaction. This process is repeated in each module Author contributions: K.B. and T.M.S. designed research; K.B. and T.M.S. performed re- until the growing peptide reaches the termination module, where it search; K.B., C.D.F., M.A.M., and T.M.S. analyzed data; and K.B. and T.M.S. wrote is elongated and then released from the NRPS, often by a thio- the paper with comments from C.D.F. and M.A.M. esterase domain. However, most NRPSs, along with their C, A, and The authors declare no conflict of interest. PCP domains, also include tailoring and/or alternative domains, This article is a PNAS Direct Submission. which cosynthetically modify the nonribosomal peptide. Data deposition: The atomic coordinates and structure factors have been deposited in the One important modification that can occur during peptide Protein Data Bank, www.pdb.org (PDB ID code 5T3E). synthesis is cyclodehydration of Cys, Ser, or Thr residues into 1To whom correspondence should be addressed. Email: [email protected]. thiazoline, oxazoline, or methyloxazoline rings, respectively, by the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NRPS heterocyclization (Cy) domain (6–12). These heterocyclic 1073/pnas.1614191114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1614191114 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 A1 PCP1 Cy2 A2 PCP2 C3 2 ATP + 2 AMP + BmdB-Cy2 Active Site. The main established signature that differ- Ala + Cys 2 PP i entiates Cy domains from C domains is an active site motif: C A1&A2 domains have a catalytic His motif of HHxxxD, and Cy domains have an Asp motif of DxxxxD (6). The first aspartate of the Asp SH SH S S adenylation + NH O O motif is essential for catalytic activity in only some Cy domains. NH2 thiolation S NH2 H2N SH When the first aspartate is mutated to alanine in HMWP2-Cy1, N AngN-Cy1, and AngN-Cy2, condensation and cyclodehydration HN C3 Cy2 Bacillamide E O condensation condensation are completely eliminated (23, 27), but in VibF-Cy1, VibF-Cy2, Tpm H2O and EpoB-Cy1, both condensation and cyclodehydration occur, although they are significantly diminished (12, 23, 28) (SI Ap- Cy2 pendix,TableS1lists current and previous Cy domain mutations). The second aspartate is critical for activity (12, 23, 27, 28), but SH S cyclodehydration SH S O O whether its role is catalytic or structural (like the aspartate at that position in C domains) (32) was not definitively determined. N S HN SH O Furthermore, a triple mutant of the Asp motif of HMWP2-Cy1 to NH2 NH introduce a C domain His motif results in a catalytically inactive 2 protein (23), demonstrating that a straight swap of the motifs is Fig. 1. Schematic representation of BmdB and the bacillamide E bio- not sufficient to interconvert catalytic activities. synthesis cycle. The A domains (orange) adenylate alanine and cysteine and In the structure of BmdB-Cy2, both aspartate residues in transfer them onto the phosphopantetheine arm of the PCP domains (blue). DxxxxD (D980 and D985) are oriented away from the active site The Cy2 domain (dark green) first catalyzes amide bond formation between (Fig. 2B). The side chain of D980 makes a bifurcated hydrogen the PCP-linked Ala and Cys residues, then catalyzes the intramolecular cyclodehydration reaction. The C3 domain (light green) catalyzes amide bond with the hydroxyl of S873 and a hydrogen bond with the bond formation between the PCP-linked Ala-thiazoline moiety and free backbone amide of L982. D985 hydrogen bonds with the sidechain tryptamine, which releases the bacillamide E from the NRPS. of S988 and the amides of A987 and F1134. The interaction this aspartate makes with a nearby arginine in C domains is present, but is water-mediated for D985-R1120 of BmdB-Cy2. Thus, these All C domain superfamily domains share the same protein fold, aspartate residues directly replace the first histidine and the as- so the overall form of the Cy domain is not in doubt (25, 26). partate in the C domain HHxxxD motif, occupying the same po- However, the features that allow the Cy domain to catalyze two sition in their respective domains and making the same or similar separate and different reactions are not known. Cy domains con- interactions with the rest of the protein (SI Appendix,Fig.S2B).