Engineering of a Nucleoside 2'-Deoxyribosyltransferase for Efficient Catalysis of 5-Halogenated-2'-Deoxypyrimidine Nucleosides via Active-Site Channel Expansion.

Nucleoside analogues play a crucial role in antiviral and anticancer drug synthesis. Owing to their high catalytic efficiency and substrate specificity, nucleoside 2'-deoxyribosyltransferases (NDTs) emerge as promising biocatalysts for nucleoside analogue synthesis. However, wild-type NDTs suffer from low yields in synthesizing halogen-substituted 2'-deoxypyrimidine nucleosides due to steric hindrance from halogen substituents in the active pocket and competitive product hydrolysis. Herein we identify an NDT from Bacillus haynesii (BhNDT) via sequence screening and introduce an E56T/D60A (BhNDT-M2) double mutation to disrupt the salt bridges and increase structural flexibility, thereby widening the active pocket and enhancing catalytic activity. The BhNDT-M2 mutant displays markedly improved activity and efficiency for synthesizing 5-fluoro-2'-deoxyuridine (FdUrd), achieving 62% of yield under 20 mM substrate, which is twice that of the wild-type. In addition, we find that high substrate concentration can bring an unexpected inhibitory effect on hydrolytic side reactions, which further improve the catalytic performance of BhNDT-M2, achieving a 55% yield of under 150 mM substrate. The BhNDT-M2 also exhibits high substrate specificity toward a range of 2'-deoxypyrimidine nucleoside analogues. To facilitate practical applications, BhNDT-M2 is immobilized onto an amino-functionalized resin, and the resulting immobilized enzyme shows excellent reusability, with only 20% loss of activity after eight cycles. Collectively, these properties establish BhNDT-M2 as a promising biocatalyst and lay the foundation for large-scale nucleoside analogue synthesis.