Abstract: The members of the Iα subfamily of aminotransferases have one of two substrate specificities: the narrow-specificity aspartate aminotransferases (AATases) and the broader-specificity tyrosine (aromatic) aminotransferases (TATases). These enzymes are well-studied, with crystal structures of six aminotransferases available and with numerous explorations of the kinetic mechanism by site-directed mutagenesis. However, we are still far from fully understanding this class of enzymes. The in vivo function in some organisms is unknown, and the mechanisms of substrate selection are unclear. Additionally, annotations of substrate specificity in sequence databases are unreliable. We investigated twelve divergent aminotransferases, and substrate specificity has been assigned for ten. The in vivo functions of these enzymes are addressed in terms of their newly assigned substrate specificities. The in vivo function of one, from Pseudomonas aeruginosa, has been determined. It is an AATase whose primary role is the biosynthesis of aspartate. The effect of active site mutations, including the A293D substitution, on substrate specificity are examined kinetically and structurally. Further protein engineering to explore substrate specificity will be enhanced by the confirmed annotations of a diverse set of aminotransferases. Such engineering can be guided by a search for groups of residues that vary with specificity. A computer program has been written to assist in this search.