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Desferrioxamine siderophores are assembled by the nonribosomal-peptide-synthetase-independent-siderophore (NIS) synthetase enzyme DesD via ATP-dependent iterative condensation of three N1-hydroxy-N1-succinyl-cadaverine (HSC) units. Current knowledge of NIS enzymology and the desferrioxamine bio-synthetic pathway does not account for the existence of most known members of this natural product family which differ in substitution patterns of the N- and C-termini. The directionality of desferrioxamine biosyn-thetic assembly, N-to-C vs C-to-N, is a longstanding knowledge gap that is limiting further progress in un-derstanding the origins of natural products in this structural family. Here, we establish the directionality of desferrioxamine biosynthesis using a chemoenzymatic approach with stable isotope incorporation and di-meric substrates. We propose a mechanism where DesD catalyzes the N-to-C condensation of HSC units to establish a unifying biosynthetic paradigm for desferrioxamine natural products in Streptomyces. 

Siderophores produced via non-ribosomal peptide synthetase (NRPS) pathways serve as critical virulence factors for many pathogenic bacteria. An improved knowledge of siderophore biosynthesis guides the development of inhibitors, vaccines, and other therapeutic strategies. Fimsbactin A is a mixed ligand siderophore derived from human pathogenic Acinetobacter baumannii that contains phenolate-oxazoline, catechol, and hydroxamate metal chelating groups branching from a central L-Ser tetrahedral unit via amide and ester linkages. Fimsbactin A is derived from two molecules of L-Ser, two molecules of 2,3-dihydroxybenzoic acid (DHB), and one molecule of L-Orn and is a product of the fbs biosynthetic operon. Here, we report the complete in vitro reconstitution of fimsbactin A biosynthesis in a cell-free system using purified enzymes. We demonstrate the conversion of L-Orn to N1-acetyl-N1-hydroxy-putrescine (ahPutr) via ordered action of FbsJ (decarboxylase), FbsI (flavin N-monooxygenase), and FbsK (N-acetyltransferase). We achieve conversion of L-Ser, DHB, and L-Orn to fimsbactin A using FbsIJK in combination with the NRPS modules FbsEFGH. We also demonstrate chemoenzymatic conversion of synthetic ahPutr to fimsbactin A using FbsEFGH and establish the substrate selectivity for the NRPS adenylation domains in FbsH (DHB) and FbsF (L-Ser). We assign a role for the type II thioesterase FbsM in producing the shunt metabolite 2-(2,3-dihydroxyphenyl)-4,5-dihydrooxazole-4-carboxylic acid (DHB-oxa) via cleavage of the corresponding thioester intermediate that is tethered to NRPS peptidyl carrier domains during biosynthetic assembly. We propose a mechanism for branching NRPS-derived peptides via amide and ester linkages via the dynamic equilibration of N-DHB-Ser and O-DHB-Ser thioester intermediates via hydrolysis of DHB-oxa thioester intermediates. We also propose a genetic signature for NRPS ‘branching’ in the presence of a terminating C-T-C motif (FbsG).