Efficient synthesis of arabinosyl nucleosides via active site engineering of theLeishmania mexicanapurine 2′-deoxyribosyltranferase

Even though 2′-deoxyribosyltransferases (NDTs) have emerged as promising and eco-friendly catalysts for synthesizing different 2’-deoxynucleoside analogs, their limited activity towards substrates featuring modifications at the 2′ -C and 3′ -C positions of the ribose moiety significantly restrict their potential application in the pharmaceutical industry. Herein we report two engineered variants from Leishmania mexicana purine deoxyribosyltransferase ( Lm PDT) with the highest reported arabinosyltransferase activity for an NDT, achieved through a semi-rational design approach. The resulting variants, Lm PDTN56L and Lm PDTN56C, display up to 13.5- and 30-fold enhanced activity on the synthesis of vidarabine compared to the wild-type enzyme. Moreover, thermal unfolding and thermal challenge experiments revealed that the improvement in arabinosyl transferase activity did not result in a stability trade-off, with Tm values similar to the wild-type enzyme and similar activity retention at 40 °C. After biochemical characterization, the optimal reaction parameters for variant Lm PDTN56C were determined to be pH 6 and a temperature interval from 30 to 45 °C. Interestingly, under optimal operational conditions (40 °C, 50 mM MES buffer pH 6) the initial 30-fold enhancement observed for Lm PDTN56C escalated to a remarkable 40-fold improvement. This comprehensive study elucidates the potential of the engineered Lm PDT variants for efficient and tailored biocatalytic synthesis of arabinosyl nucleosides, paving the way for enhanced applications in nucleoside analog production.