Nicotianamine synthase (NAS) catalyzes the biosynthesis of the low-molecular-mass metal chelator nicotianamine (NA) from the 2-aminobutyrate moieties of threeSAMmolecules. NA has central roles in metal nutrition and metal homeostasis of flowering plants. The enzymatic function of NAS remains poorly understood. Crystal structures are available for archaeal and bacterial NAS-like proteins that carry out simpler aminobutanoyl transferase reactions. Here, we report amino acids essential for the activity of AtNAS1 based on structural modeling and site-directed mutagenesis. Using a newly developed enzyme-coupled continuous activity assay, we compare differing NAS proteins identified through multiple sequence alignments and phylogenetic analyses. In most NAS of dicotyledonous and monocotyledonous plants (class Ia and Ib), the core-NAS domain is fused to a variable C-terminal domain. Compared to fungal and moss NAS that comprise merely a core-NAS domain (class III), NA biosynthetic activities of the four paralogousArabidopsis thalianaNAS proteins were far lower. C-terminally trimmed core-AtNAS variants exhibited strongly elevated activities. Of 320 amino acids of AtNAS1, twelve, 287-TRGCMFMPCNCS-298, accounted for the autoinhibitory effect of the C terminus, of which approximately one-third was attributed to N296 within a CNCS motif that is fully conserved in Arabidopsis. No detectable NA biosynthesis was mediated by two representative plant NAS proteins that naturally lack the C-terminal domain, class IaArabidopsis halleriNAS5 andMedicago truncatulaNAS2 of class II which is found in dicots and diverged early during the evolution of flowering plants. Next, we will address a possible posttranslational release of autoinhibition in class I NAS proteins.