Monobactam formation in sulfazecin by a nonribosomal peptide synthetase thioesterase

Abstract

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

Structure of a bound peptide phosphonate reveals the mechanism of nocardicin bifunctional thioesterase epimerase-hydrolase half-reactions

“…While the canonical biosynthesis carried out by NRPSs has been well-studied, an increasing number of examples exist in which NRPS domains are shown to catalyze unusual activities 4 . These activities include amide formation by adenylation domains 5,6 , condensation domain-catalyzed amide formation between a propionate and a pteridine ring in an unusual “pepteridine” natural product 7 , thioesterase domain-mediated condensation of two α-keto carboxylates in the production of quinone or furanone derivatives 8,9 , β-lactone and β-lactam cyclization by thioesterase domains 10–12 , and a Dieckmann condensation catalyzed by an unusual reductase domain, which lacks the conventional catalytic triad, involved in the release of cyclopiazonate tetramic acid neurotoxin 13 . Some of these activities reflect more limited changes in the overall synthetic program, for example, the use of an alternate nucleophile to attack the adenylate directly in formation of the amide bond that bypasses the PCP-bound thioester intermediate.…”

“…To complement and further interpret a body of mechanistic studies 10,15,17,18 to understand the unusual steps in nocardicin A biosynthesis, we present structures of this dual-function thioesterase domain NocTE in the unliganded and peptide-bound states. Structural studies of NRPS catalytic domains benefit from the availability of ligands or ligand analogs to provide insight into the active site architecture and catalytic mechanism 19 .…”

Constructing and deconstructing the bacterial cell wall

“…294,295 Moreover, the operon for bulgecin biosynthesis in the bacterium Paraburkholderia acidophilia is contiguous 296 with that of the monobactamclass β-lactam, sulfazecin. [297][298][299] The presumption that P. acidophilia coordinates the biosynthetic production of the bulgecins with sulfazecin was implicitly validated by the studies of Imada et al 300 and our own recent findings. 301 More recently we documented selectivity in inhibition of the members of the LT family.…”

“…The molecular targets of the bulgecins—their structure (Figure ) is recognizable as characteristic of iminosaccharide‐type inhibitors of glycoside hydrolases—were the LT enzymes . Moreover, the operon for bulgecin biosynthesis in the bacterium Paraburkholderia acidophilia is contiguous with that of the monobactam‐class β‐lactam, sulfazecin . The presumption that P. acidophilia coordinates the biosynthetic production of the bulgecins with sulfazecin was implicitly validated by the studies of Imada et al and our own recent findings .…”

Refining and expanding nonribosomal peptide synthetase function and mechanism

“…This and additional biochemical data supported the role of the TE as the β-lactone forming enzyme, first catalyzing transacylation of the dipeptide precursor from the CP domain to the Cys of the TE domain, then catalyzing intramolecular attack to generate a strained β-lactone ring ( Figure 12B). Shortly after the discovery of TE-catalyzed β-lactone formation, the mechanistic details regarding the biosynthesis of a comparable β-lactam ring of the tripeptide sulfazecin were reported ( Figure 12A) [76]. The same Ser-to-Cys substitution was also found within the TE domain of the terminal module of the NRPS SulM.…”