Structure-guided mining and engineering of nicotinamide riboside kinase for efficient synthesis of nicotinamide mononucleotide

Nicotinamide mononucleotide (NMN) is a bioactive precursor to nicotinamide adenine dinucleotide (NAD⁺) and has demonstrated anti-aging activity. Nicotinamide riboside kinase (NRK) can convert nicotinamide ribose (NR) into NMN in one step, but NRKs with high catalytic efficiency are rare and are difficult to engineer. In this study, two novel NRKs from Sparassis crispa and Beauveria bassiana were identified. They displayed impressive specific activities of 6.14 U/mg and 6.66 U/mg with high substrate (NR) conversion rates. Furthermore, structural modelling analysis and semi-rational design were used to develop variants of BEANRK. Variant M1 (V172K) exhibited a 1.2-fold of the wild-type BEANRK (WT) in enzyme activity and 91 % of substrate conversion rate with improved thermal stability compared to WT. Moreover, variants generated from site-specific mutagenesis were also computationally analyzed to elucidate which interactions between key amino residues and substrates facilitated an increase in catalytic activity and conversion rate. This study not only identified new NRKs but also provided insights into which amino acid residues were pivotal to the catalytic activity of NRK, paving the way for future NRK engineering to improve the industrial production of NMN.