The structure of the monobactam-producing thioesterase domain of SulM forms a unique complex with the upstream carrier protein domain

Abstract

Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain that catalyzes an unusual β-lactam forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the β-lactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The structure illustrates a constrained active site that orients the substrate properly for β-lactam formation. In this regard, the structure is similar to the β-lactone forming thioesterase domain responsible for the production of obafluorin. Analysis of the structure identifies features that are responsible for this four-membered ring closure and enable bioinformatic analysis to identify additional, uncharacterized β-lactam-forming biosynthetic gene clusters by genome mining.

Keywords: NRPS; Nonribosomal Peptide Synthetase; thioesterase; β-lactam antibiotic.

Figure 1.. Biosynthesis of Sulfazecin.

A. Two NRPS proteins, SulI and SulM, harbor three modules necessary to produce the desmethoxysulfazecin. The SulI module contains an adenylylsulfate kinase, responsible for production of PAPS, as well as the initiation module for D-glutamate. SulM contains two modules that incorporate L-alanine, which is epimerized to the D- stereoisomer, in module 2 and L-DAP in module 3. B. The final steps in sulfazecin production include the in trans sulfonation catalyzed by SulN, and then β-lactam formation by the thioesterase domain, resulting in the production of desmethoxysulfazecin. Two final steps, catalyzed by the nonheme iron oxygenase SulO and methyltransferase SulP produce sulfazecin.

Figure 2.. Structure of SulM thioesterase domain.

The SulM thioesterase domain adopts a conventional α/β hydrolase fold with a central β-sheet surrounded on both sides by α-helices. A. The catalytic triad illustrates a nucleophilic cysteine residue Cys2818 that is positioned on the loop following strand β5, interacting with His2956 and Asp2926. B. The active site pocket is formed from residues on the lid loops and lid helices. C. SulM and ObiF thioesterase domains contain a catalytic triad in which the aspartic acid is positioned on the loop following strand β7. In contrast, the aspartic acid residue of Vlm2, like all other structurally characterized NRPS thioesterase domains, is positioned in the loop following strand β6.

Figure 3.

Examination of monobactam formation in SulTE. A. γ-D-Glu-D-Ala-L-Dap linked to the panethiene arm of PCP₃ in the NRPS SulM is sulfonated in trans to the corresponding sulfamate and delivered to the SulTE catalytic Cys2818 by transthioesterification and cyclized to the monocyclic β-lactam product and released. Subsequent oxidation by SulO and O-methylatioin by SulP yield the monobactam sulfazecin. (B) The C-terminal amino acid of the tripeptide in A was replaced with L-Glu to mimic the distal charge of the native sulfamate.

Figure 4.. Pantetheine tunnel of the SulM thioesterase active site.

The SulM thioesterase domain binds the PCP, allowing the pantetheine to pass through a mostly hydrophobic tunnel to enter the active site near the catalytic cysteine Cys2818, which has been mutated to an alanine for the complex structure.

Figure 5:. Comparison of SulM_PCP-TE and EntF_PCP-TE interfaces.

The structures of the PCP-thioesterase didomain complexes were superimposed on the basis of the thioesterase domain, excluding the dynamic lid loops. The holo-PCP domains of each complex are represented in pink (SulM) or yellow (EntF). The PCP domains bind in the same region but adopt a different orientation. The α2 helix of each PCP is depicted with an arrow that is colored red to green in the N- to C- direction. The angle between the two helices is 50°, depicting the alternate positions adopted by the PCP domains.

Figure 6.. The thioesterase domain creates a cavity for β-lactam/β-lactone formation.

Multistep reaction catalyzed by A) the SulM thioesterase and B) ObiF1 thioesterase domain. C. Active site of the thioesterase domain with a modeled linear sulfazecin peptide that cradles the substrate for β-lactam formation. Shown in pink is the pantetheine arm observed in the SulM PCP-TE structure. D. Active site of the thioesterase domain of ObiF1 (PDB 6N9E) that positions the modeled obafluorin peptide for β-lactone formation. The view in panel D is rotated slightly around the Y-axis relative to panel C.

Figure 7.. Anti-SMASH analysis of likely new beta-lactam forming clusters.

Anti-SMASH 7.0 was used to search for biosynthetic clusters (BGCs) using SulTE sequence as query in PSI-BLAST. The BGCs harboring SulTE-like domains were analyzed for module architecture and predicted substrates for each module. The predicted adenylation domain substrates differed from Sulfazecin cluster except in last module which activates DAP required for β-lactam ring formation. Accession code for each cluster is provided in brackets and for each protein on the arrow. Predicted product and Stachelhaus code residues for adenylation domain in last module are shown in supplemental table S3. Figure legends are as followed, A: adenylation domain, CP: carrier protein, C: condensation domain, C in gray: probable condensation domain, E: epimerization domain, TE: thioesterase domain, nMT: methyltransferase domain, ?: FkbH-like domain, S: sulfotransferase domain.