Nicotinamide 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 V190S/L336Q mutant displayed a 4-fold 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 work highlights the catalytic advantage of intramolecular substrate channeling and presents a generalizable strategy for enhancing the performance of multifunctional biocatalysts.