Mechanistic and Structural Analyses of Non-Heme Iron Enzyme TqaM for α-Tertiary Amino Acid Synthesis

α-Tertiary amino acids (ATAAs) are versatile building blocks for the synthesis of biologically active compounds. Although synthetic and enzymatic approaches to ATAA synthesis have been developed, additional methods for ATAA production remain in high demand. Here, we report detailed mechanistic and structural analyses of TqaM, a non-heme iron-dependent oxygenase that catalyzes the key oxidative decarboxylation of a β-amino acid to generate 2-aminoisobutyric acid, a representative ATAA. <i>In vitro</i> analyses revealed that TqaM strictly recognizes the stereochemistry at the C2 position of the substrate and initiates the reaction by abstracting a C2-hydrogen atom with one equivalent of molecular oxygen. Structural analysis and site-directed mutagenesis suggested that an active site histidine residue functions as an acid/base catalyst and the N-terminal loop of the enzyme plays a critical role in substrate selectivity. Finally, we demonstrate TqaM's remarkable substrate promiscuity toward α-hydroxy-β-amino acids, enabling the efficient synthesis of diverse ATAAs with potential biocatalytic applications.