Structure and Mechanism of the Siderophore-Interacting Protein from the Fuscachelin Gene Cluster of <i>Thermobifida fusca</i>

Microbial iron acquisition is a complex process and frequently a key and necessary step for survival. Among the several paths for iron assimilation, small molecule siderophore-mediated transport is a commonly employed strategy of many microorganisms. The chemistry and biology of the extraordinary tight and specific binding of siderophores to metal is also exploited in therapeutic treatments for microbial virulence and metal toxicity. The intracellular fate of iron acquired via the siderophore pathway is one of the least understood steps in the complex process at the molecular level. A common route to cellular incorporation is the single-electron reduction of ferric to ferrous iron catalyzed by specific and/or nonspecific reducing agents. The biosynthetic gene clusters for siderophores often contain representatives of one or two families of redox-active enzymes: the flavin-containing “siderophore-interacting protein” and iron–sulfur ferric siderophore reductases. Here we present the structure and characterization of the siderophore-interacting protein, FscN, from the fuscachelin siderophore gene cluster of <i>Thermobifida fusca</i>. The structure shows a flavoreductase fold with a noncovalently bound FAD cofactor along with an unexpected metal bound adjacent to the flavin site. We demonstrated that FscN is redox-active and measured the binding and reduction of ferric fuscachelin. This work provides a structural basis for the activity of a siderophore-interacting protein and further insight into the complex and important process of iron acquisition and utilization

Interdomain and Intermodule Organization in Epimerization Domain Containing Nonribosomal Peptide Synthetases

Nonribosomal peptide synthetases are large, complex multidomain enzymes responsible for the biosynthesis of a wide range of peptidic natural products. Inherent to synthetase chemistry is the thioester templated mechanism that relies on protein/protein interactions and interdomain dynamics. Several questions related to structure and mechanism remain to be addressed, including the incorporation of accessory domains and intermodule interactions. The inclusion of nonproteinogenic d-amino acids into peptide frameworks is a common and important modification for bioactive nonribosomal peptides. Epimerization domains, embedded in nonribosomal peptide synthetases assembly lines, catalyze the l- to d-amino acid conversion. Here we report the structure of the epimerization domain/peptidyl carrier protein didomain construct from the first module of the cyclic peptide antibiotic gramicidin synthetase. Both <i>holo</i> (phosphopantethiene post-translationally modified) and <i>apo</i> structures were determined, each representing catalytically relevant conformations of the two domains. The structures provide insight into domain–domain recognition, substrate delivery during the assembly line process, in addition to the structural organization of homologous condensation domains, canonical players in all synthetase modules