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The subfamily Iα aminotransferases are typically categorized as having narrow specificity toward carboxylic amino acids (AATases), or broad specificity that includes aromatic amino acid substrates (TATases). Because of their general role in central metabolism and, more specifically, their association with liver-related diseases in humans, this subfamily is biologically interesting. The substrate specificities for only a few members of this subfamily have been reported, and the reliable prediction of substrate specificity from protein sequence has remained elusive. In this study, a diverse set of aminotransferases was chosen for characterization based on a scoring system that measures the sequence divergence of the active site. The enzymes that were experimentally characterized include both narrow-specificity AATases and broad-specificity TATases, as well as AATases with broader-specificity and TATases with narrower-specificity than the previously known family members. Molecular function and phylogenetic analyses underscored the complexity of this family's evolution as the TATase function does not follow a single evolutionary thread, but rather appears independently multiple times during the evolution of the subfamily. The additional functional characterizations described in this article, alongside a detailed sequence and phylogenetic analysis, provide some novel clues to understanding the evolutionary mechanisms at work in this family.