Site-Selective Deuteration of Amino Acids through Dual-Protein Catalysis

Deuterated amino acids have been recognized for their utility in drug development, for facilitating nuclear magnetic resonance (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 hydrogen-deuterium (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 ultraviolet (UV)-vis spectroscopy to probe the mechanism of a two-protein system responsible for the biosynthesis of L-allo-Ile. We show that an aminotransferase (DsaD) can pair with a small partner protein (DsaE) to catalyze C alpha and C beta H/D exchange of amino acids, while reactions without DsaE lead exclusively to C alpha-deuteration. With conditions for improved catalysis, we evaluate the substrate scope for C alpha/C beta-deuteration and demonstrate the utility of this system for preparative-scale, selective labeling of amino acids.

Automated summary

Deuterated amino acids play important roles in drug development, nuclear magnetic resonance (NMR) analysis, and enzyme mechanism studies. Enzymatic methods for introducing deuterium offer improved efficiency compared to small molecule-based methods. However, achieving site selectivity for enzyme-mediated deuteration remains a challenge.