The pvcABCD operon of Pseudomonas aeruginosa encodes four proteins (PA2254, PA2255, PA2256, and PA2257) that form a cluster that is responsible for the synthesis of a cyclized isocyano derivative of tyrosine. These proteins, which were identified originally as being responsible for a step in the maturation of the chromophore of the peptide siderophore pyoverdine, have been identified recently as belonging to a family of proteins that produce small organic isonitriles. We report that strains harboring a disruption in the pvcA or pvcB genes are able to grow in iron-depleted conditions and to produce pyoverdine. Additionally, we have determined the three-dimensional crystal structures of PvcA and PvcB. The structure of PvcA demonstrates a novel enzyme architecture that is built upon a Rossmann fold. We have analyzed the sequence conservation of enzymes within this family and identified six conserved motifs. These regions of the protein cluster around a putative active site cavity. The structure of the PvcB protein confirms it is a member of the Fe2+/alpha-ketoglutarate-dependent oxygenase family of enzymes. The active site of PvcB is compared to the structures of other family members and suggests that a conformational change to order several loops will accompany the binding of ligands.