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

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 α-keto acids. Additionally, hKYAT1 is known to catalyze the β-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 13 mutant variants was systematically evaluated against sixteen different amino acid substrates, including kynurenine, selenomethionine (SeMet), and Se-methylselenocysteine (MSC), as well as for the β-elimination of SeMet and MSC. Our results demonstrate that mutations of residues E27 in the catalytic site and H279 in the substratestabilizing site significantly enhanced the transamination of several amino acids, including phenylalanine, tryptophan, histidine, and MSC. The H279F mutation increased transamination and β-elimination of MSC by 2- and 1.5-fold, respectively. Furthermore, mutation at the ligand-binding residues R398, F125, and N185 substantially reduced MSC transamination activity of hKYAT1. Interestingly, none of the tested mutations affected the transamination of l-kynurenine, a natural substrate of hKYAT1. Altogether, these findings support future investigation into hKYAT1 as a modifiable target in selenium-mediated anticancer approaches.