Bacterial pathogenesis frequently requires metal acquisition by specialized, small-molecule metallophores. We hypothesized that the Gram-negative Pseudomonas aeruginosa encodes the enzymes nicotianamine synthase (NAS) and opine dehydrogenase (ODH), biosynthesizing a new class of opine metallophore, previously characterized only in the unrelated Gram-positive organism Staphylococcus aureus. The identity of this metallophore, herein named pseudopaline, was determined through measurements of binding affinity, the in vitro reconstitution of the biosynthetic pathway to screen potential substrates, and the confirmation of product formation by mass spectrometry. Pseudopaline and the S. aureus metallophore staphylopine exhibit opposite stereochemistry for the histidine moiety, indicating unique recognition by NAS. Additionally, we demonstrate SaODH catalysis in the presence of pyruvate, as previously shown, but also oxaloacetate, suggesting the potential for the production of a variant form of staphylopine, while PaODH specifically recognizes α-ketoglutarate. Both the staphylopine and pseudopaline operons have been implicated in the pathogenesis of key infectious disease states and warrant further study.