Engineering the Activity and Stability of dCMP Deaminase To Achieve Efficient and Simple Production of Uridine...

Uridine monophosphate (UMP) is an essential nucleotide for infant formula, drug precursors, and DNA synthesis, yet current production methods face challenging downstream purification. Here, we established an efficient biocatalytic system for direct cytidine monophosphate (CMP)-to-UMP conversion using the deoxycytidine monophosphate deaminase (DCTD) from Enterobacteriaceae phage PG7, naturally active toward CMP. Evolutionary Scale Modeling-guided surface mutations combined with high-throughput screening generated the L99I variant, enhancing catalytic efficiency by 50-fold. Subsequent optimization through ProteinMPNN-guided design and ancestral sequence reconstruction produced the L99I/I92K double mutant, exhibiting 8-fold improved thermal stability and a total turnover number of 122,000. Molecular dynamics simulations elucidated the structural basis of the enhanced activity and stability. Using this optimal variant, 500 mM CMP was completely converted to UMP in 3 h with 99% yield. This study demonstrates a practical, green strategy for UMP production and highlights how rational enzyme engineering informed by allosteric insights can simultaneously improve catalytic efficiency, substrate specificity, and industrial applicability.