Site-selective deuteration of amino acids through dual protein catalysis

Deuterated amino acids have been recognized for their utility in drug development, for facilitating NMR analysis, and as probes for enzyme mechanism. Small molecule-based methods for the site-selective synthesis of deuterated amino acids typically involve de novo synthesis of the compound from deuterated precursors. In comparison, enzymatic methods for introducing deuterium offer improved efficiency, operating directly on free amino acids to achieve hydrogendeuterium (H/D) exchange. However, site-selectivity remains a significant challenge for enzyme-mediated deuteration, limiting access to desirable deuteration motifs. Here we use enzyme-catalyzed deuteration, combined with steady-state kinetic analysis and UV-vis spectroscopy to probe the mechanism of a two-protein system responsible for the biosynthesis of L-alloIle. We show an aminotransferase (DsaD) can pair with a small partner protein (DsaE) to catalyze Cα and Cβ H/D exchange of amino acids, while reactions without DsaE lead exclusively to Cα-deuteration. With conditions for improved catalysis, we evaluate the substrate scope for Cα/Cβ-deuteration and demonstrate the utility of this system for preparative-scale, selective labeling of amino acids.