Molecular function prediction for a family exhibiting evolutionary tendencies toward substrate specificity swapping: Recurrence of tyrosine aminotransferase activity in the Iα subfamily

Abstract

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.

Aspartate aminotransferase: An old dog teaches new tricks

“…The AAT-to-TAT conversion was chosen because of the excellent work from Kirsch’s laboratory on the same conversion, which provides an important benchmark for comparison. [92–97]…”

Structural basis for the relaxed substrate selectivity of Leishmania mexicana broad specificity aminotransferase

“…Here, a ratio of k cat /K m for aspartate/2-oxoglutarate versus k cat /K m for phenylalanine/2-oxoglutarate of more than 1 indicates specificity for asparate. In an attempt to predict substrate specificity, Muratore et al used amino acid conservation around the cofactor to identify ten subgroups within the I␣-subfamily and assessed substrate preference for one member of each subgroup [20]. This revealed that predictions are difficult because closely related subgroups showed opposite specificity.…”

COMBREX-DB: an experiment centered database of protein function: knowledge, predictions and knowledge gaps

“…In particular, we included BLAST style methods, phylogenetic methods ( 21 ), general functional linkages stored in VISANT that are computed based on a variety of context methods ( 22 – 24 ). Future versions of COMBREX aim to allow user-selected methods of organizing genes, whether using clusters, or functional linkage networks based on ideas described in ( 24 – 27 ).…”