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

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 of SulM, which catalyzes an unusual ß-lactam-forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam 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 position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for ß-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the ß-lactone-forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized ß-lactam-forming biosynthetic gene clusters by genome mining.

Monobactam formation in sulfazecin by a nonribosomal peptide synthetase thioesterase.

The N-sulfonated monocyclic ß-lactam ring characteristic of the monobactams confers resistance to zinc metallo-ß-lactamases and affords the most effective class to combat carbapenem-resistant enterobacteria (CRE). Here we report unprecedented nonribosomal peptide synthetase activities, wherein an assembled tripeptide is N-sulfonated in trans before direct synthesis of the ß-lactam ring in a noncanonical, cysteine-containing thioesterase domain. This means of azetidinone synthesis is distinct from the three others known in nature.

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

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 ß-lactam forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the ß-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 ß-lactam formation. In this regard, the structure is similar to the ß-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 ß-lactam-forming biosynthetic gene clusters by genome mining.

Identification and Characterization of the Sulfazecin Monobactam Biosynthetic Gene Cluster.

The monobactams, exemplified by the natural product sulfazecin, are the only class of ß-lactam antibiotics not inactivated by metallo-ß-lactamases, which confer bacteria with extended-spectrum ß-lactam resistance. We screened a transposon mutagenesis library from Pseudomonas acidophila ATCC 31363 and isolated a sulfazecin-deficient mutant that revealed a gene cluster encoding two non-ribosomal peptide synthetases (NRPSs), a methyltransferase, a sulfotransferase, and a dioxygenase. Three modules and an aberrant C-terminal thioesterase (TE) domain are distributed across the two NRPSs. Biochemical examination of the adenylation (A) domains provided evidence that L-2,3-diaminopropionate, not L-serine as previously thought, is the direct source of the ß-lactam ring of sulfazecin. ATP/PPi exchange assay also revealed an unusual substrate selectivity shift of one A domain when expressed with or without the immediately upstream condensation domain. Gene inactivation analysis defined a cluster of 13 open reading frames sufficient for sulfazecin production, precursor synthesis, self-resistance, and regulation. The identification of a key intermediate supported a proposed NRPS-mediated mechanism of sulfazecin biosynthesis and ß-lactam ring formation distinct from the nocardicins, another NRPS-derived subclass of monocyclic ß-lactam. These findings will serve as the basis for further biosynthetic research and potential engineering of these important antibiotics.

Whole-Genome Shotgun Sequencing of Two ß-Proteobacterial Species in Search of the Bulgecin Biosynthetic Cluster.

We have produced draft whole-genome sequences for two bacterial strains reported to produce the bulgecins as well as NRPS-derived monobactam ß-lactam antibiotics. We propose classification of ATCC 31363 as Paraburkholderia acidophila. We further reaffirm that ATCC 31433 (Burkholderia ubonensis subsp. mesacidophila) is a taxonomically distinct producer of bulgecins with notable gene regions shared with Paraburkholderia acidophila. We use RAST multiple-gene comparison and MASH distancing with published genomes to order the draft contigs and identify unique gene regions for characterization. Forty-eight natural-product gene clusters are presented from PATRIC (RASTtk) and antiSMASH annotations. We present evidence that the 10 genes that follow the sulfazecin and isosulfazecin pathways in both species are likely involved in bulgecin A biosynthesis.