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ABSTRACTSNicotinamide adenine dinucleotide (NAD?) is an essential redox cofactor widely used in industrial biocatalysis and therapeutic applications. Conventional chemical synthesis of NAD? is hazardous and inefficient, while enzymatic approaches, particularly dual?enzyme cascades involving nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), suffer from low catalytic efficiency due to spatially separated active sites and poor intermediate channeling. Here, we report a direct, one?step NAD? biosynthetic route using Haemophilus influenzae NadR, a naturally bifunctional enzyme that naturally integrates NRK and NMNAT activities into a single polypeptide chain. To address the rate?limiting NR phosphorylation step, we developed a loop engineering–docking combinatorial simulation (LEDCS) strategy to rationally redesign the NRK domain. The resulting V241S/L387Q mutant displayed a fourfold increase in enzymatic activity and a 2.4?fold improvement in catalytic efficiency relative to the wild type. Structural and interaction analyses revealed that the P?loop plays a critical role in coordinating NR and ATP binding, while enhanced hydrophilicity in the substrate?binding pocket improved substrate affinity. This study highlights the catalytic advantage of intramolecular substrate channeling and presents a generalizable strategy for enhancing the performance of multifunctional biocatalysts.