Structure Guided Engineering of Bifunction Enzyme NadR for Enhanced Nicotinamide Adenine Dinucleotide Production
Year: 2025
Type: article
Abstract: 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 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.
Source: Biotechnology and Bioengineering
DOI: 10.1002/bit.70097
Authors: Dianju Wei, Yinfeng Huang, Daifu Shi, Weibin Shu, Shuping Zou, Yaping Xue, Yuguo Zheng
Institution: Zhejiang University of Technology
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $5,060
OpenAlex ID: W4415905282