Structure‐Guided Engineering of a Short‐Chain Dehydrogenase LfSDR1 for Efficient Biosynthesis of (R)‐9‐(2‐Hydroxypropyl)adenine, the Key Intermediate of Tenofovir

(R)‐9‐(2‐hydroxypropyl) adenine ((R)‐HPA) is an important intermediate for the synthesis of tenofovir and its prodrugs. Herein, structure‐guided rational design of short‐chain dehydrogenase LfSDR1 was adopted to improve the catalytic performance for enantioselective synthesis of (R)‐HPA at high substrate loading. The crystal structures of LfSDR1 in its apo form as well as in complex with NADPH were solved, which were used for mutagenesis studies and illustration mechanism. Three residues (G92, E141 and V186) were identified as hotspots by structural analysis, and variants V186A/G92V and V186A/G92V/E141L with remarkably improved activity were obtained. By molecular dynamics (MD) simulation of WT and variants, G92V plays a key role in enzyme‐substrate interaction in the binding pocket. Whole cells expressing the mutant LfSDR1‐V186A/G92V and glucose dehydrogenase BsGDH from Bacillus subtilis were used as the catalyst, and up to 200 g L⁻¹ substrate without cosolvent was completely converted to (R)‐HPA with 99.9% ee and a high space‐time yield (STY) of 800 g L⁻¹ day⁻¹. This study improves the understanding of the catalytic mechanism of LfSDR and provides a potential biocatalytic strategy for industrial synthesis of (R)‐HPA.