Fimsbactin A, a siderophore produced by Acinetobacter baumannii, contains phenolate-oxazoline, catechol, and hydroxamate metal chelation groups that branch from the α-carbon of a central L-serine unit. Our lab has previously reconstituted the fimsbactin biosynthetic pathway through in vitro enzyme assays with a four-subunit, eleven-domain nonribosomal peptide synthetases (NRPSs) system (FbsH, FbsE, FbsF, and FbsG). However, the programming rule of the NRPS modules required to achieve the branched peptide architecture of the fimsbactin scaffold remains unsolved. The presence of a truncated adenylation (A) domain, tandem cyclization (Cy) domains, and the disproportionate number of peptidyl-carrier-protein (PCP) domains in this pathway suggests a high degree of non-collinearity. The terminal module FbsG also employs an unusual domain architecture consist of N-terminal condensation (C1), peptidyl-carrier-protein, and C-terminal condensation (C2) domains. Here, we utilize site-specific mutagenesis to disable each PCP, Cy, and C domain to provide snapshots of individual biosynthetic steps during catalysis. We have found that the A domain in FbsE can iteratively aminoacylates the PCP domains in FbsE and FbsG. We confirm that the C1 domain is catalytically inactive. We also observe a high tolerance of mutations on the Cy domain in FbsE towards product formation, while the mutation on the Cy domain in FbsF nearly abolishes product formation. As the FbsE Cy-domain is largely redundant, we hypothesize that the FbsF Cy domain is multi-functional and catalyzes: 1) both amide and ester bond formation, 2) cyclodehydration, and 3) a second chain elongation event to yield the branched peptidyl intermediate. Together, our study presents a new biosynthetic paradigm for nonribosomal peptides that branch through amide- and/or ester-bond linkages with the unusual domain architecture of the FbsEFG NPRS modules.

Branched sderophore biosynthesis catalyzed by non-canonical nonribosomal peptide synthetases

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