Project Summary/Abstract Opine metallophores are recently identified novel metal acquisition small molecules made by diverse bacterial pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, and Yersinia pestis. The operons for biosynthesis, export and re-uptake have only been reported in the last two years, and yet these systems have already been shown to be linked to pathogenesis in mouse models for bacteremia, lung infections and burn wound infections. The biosynthesis of these metallophores is especially intriguing, because they are not made by the well-studied nonribosomal peptide synthetases or polyketide synthases. Instead, these pathogens have evolved a biosynthetic system that is dependent on a nicotianamine synthase and an opine dehydrogenase. Nicotianamine synthases (NAS) have been primarily studied in plants, and iteratively join the aminobutyrate moieties of S-adenosylmethionine. Opine dehydrogenases (ODH) are made by plant pathogens and molluscs, and generate linkages between an amino acid and an α-keto acid. Neither class of enzymes in well-studied, so we propose here fill to this gap-in-knowledge, providing a structural biology and mechanistic enzymology characterization of these enzymes. We will also provide the first structural characterization of the metallophores themselves. This basic science research will lay the necessary foundation for future novel antimicrobial drug design efforts.