Developing High Performance N-Oxygenase for Azomycin Synthesis through Ancestral Sequence Reconstruction

Azomycin, as a representative example of valued nitro compounds, suffers from harsh reaction conditions and low yields in its traditional synthesis methods. Biocatalytic synthesis using N-oxygenase offers a green alternative but is constrained by the enzyme's limited efficiency and stability. To overcome these limitations, this study employed ancestral sequence reconstruction to identify a high-performance N-oxygenase. Seven ancestral node proteins, inferred from 83 homologous sequences, were resurrected and screened. The derived enzyme ASR-90 exhibited superior catalytic efficiency (k_cat/K_m) toward 2-aminoimidazole, exceeding its parent enzyme and a key mutant by 546-fold and 92-fold, respectively. Furthermore, ASR-90 exhibited improved acid tolerance (optimal pH 4.5) and a 4-fold longer half-life at 50 °C. Under optimal conditions, it achieved 50.9% substrate conversion in 20 min─2.7-fold higher than that of the best reported enzyme. The substrate scope analysis revealed that ASR-90 displayed catalytic ability on 16 non-natural substrates. Molecular dynamics simulations indicated that the enhanced catalytic efficiency stems from increased substrate binding stability and affinity. Its broad substrate scope and preference for aromatic amines are attributed to a balanced active-site pocket, electrostatic guidance, and a continuous aromatic track within the substrate channel. This study establishes ancestral sequence reconstruction as an effective strategy for developing robust N-oxygenases, facilitating the green biosynthesis of azomycin and its derivatives.