Mutational insights into human kynurenine aminotransferase 1: modulation of transamination and β-elimination activities across diverse substrates

Abstract

Human kynurenine aminotransferase 1 (hKYAT1) plays a crucial role in the transamination of aromatic amino acids and kynurenine. This promiscuous homodimeric enzyme transaminates various amino acids into their corresponding alpha-keto acids. Additionally, hKYAT1 is known to catalyze the beta-elimination of cysteine-S-conjugates and cysteine-Se-conjugates. In this study, we performed mutational analyses of hKYAT1, targeting its catalytic, ligand-binding, and substrate-binding sites. The transamination activity of thirteen mutant variants was systematically evaluated against sixteen different amino acid substrates, including kynurenine, selenomethionine (SeMet), and Se-methylselenocysteine (MSC), as well as for the {beta}-elimination of SeMet and MSC. Our results demonstrate that mutations of residues E27 in the catalytic site and H279 in the substrate-stabilizing site significantly enhanced the transamination of several amino acids, including phenylalanine, tryptophan, histidine, and MSC. The H279F mutation increased transamination and {beta}-elimination of MSC by two- and 1.5-fold, respectively. Furthermore, mutation at the ligand-binding residues R398 and N185 abolished the transamination activity of hKYAT1. Interestingly, none of the tested mutations affected the transamination of L-kynurenine, a natural substrate of hKYAT1. These findings provide a foundation for the rational design of selective inhibitors with potential therapeutic applications.