# Advances in the Synthesis and Physiological Metabolic Regulation of Nicotinamide Mononucleotide
## 3 Biosynthesis of NMN
### Enzymes Catalyzing the Synthesis of NMN  
Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the synthesis of NMN from nicotinamide (NAM) and phosphoribosyl pyrophosphate (PRPP). In mammalian cells, NAMPT is the rate-limiting enzyme of the NAD\({}^{+}\) metabolic pathway. [PERSON] et al. [28] discovered that NAMPT exhibited 46-fold lower efficiency compared to nicotinamide mononucleotide adenosine transferase (NMNAT), another enzyme involved in the salvage pathway, indicating that the reaction catalyzed by NAMPT represents the bottleneck of NAD\({}^{+}\) synthesis from nicotinamide. [PERSON] et al. [29] individually expressed NAMPT orthologs from _Mus musculus_, _Shewanella oneidensis_, and _Haemophilus ducreyi_ in _E. coli_. Compared to the two bacterial NAMPT enzymes, the mammalian NAMPT from _Mus musculus_ exhibited a relatively lower yield of NMN in _E. coli_. Therefore, it is crucial to identify NAMPT variants with high enzyme activity for efficient industrial production of NMN by screening diverse sources. [PERSON] et al. [30] compared the heterologous expression of 10 different NAMPT genes from mammalian and bacterial sources in _E. coli_ and found that the bacterial NAMPT gene from _Chitinophaga pinensis_ exhibited the highest expression efficiency in _E. coli_, with a 2.4-fold higher enzyme activity than that of NAMPT from _S. oneidensis_. Similarly, [PERSON] et al. [12] selected eight previously reported NAMPT genes from various sources for expression in _E. coli_ and demonstrated that the NAMPT from _Vibrio_ bacteriophage KVP40 was capable of intracellularly synthesizing 81.3 mg/L NMN using 200 mg/L nicotinamide as substrate, representing a significant increase in extracellular NMN accumulation. Nicotinamide N-methyltransferasearse is a crucial cytosolic protein and a key methyltransferase. Inside cells, it catalyzes the transfer of a methyl group from the cofactor S-adenosyl methionine (SAM) to nicotinamide, leading to the generation of N1-methylnicotinamide (MNA) and S-adenosylyl-L-homocysteine (SAH) [31; 32]. In this case, NNMT exerts its influence on both the NAD\({}^{+}\) salvage pathway and the methionine cycle by concurrently depleting both NAM and SAM. As a crucial precursor for NMN synthesis, NAM cannot replenish the NAD\({}^{+}\) salvage pathway and regulate NAD\({}^{+}\) levels upon methylation. Studies  
Figure 5: AMP acid hydrolysis method. have demonstrated a significant impact of NNMT activity on cellular NAD\({}^{+}\) content. Therefore, reducing NNMT activity using inhibitors represents a promising strategy to alleviate NAM depletion and enhance NAD\({}^{+}\) availability [33; 34; 35]. NNMT has been demonstrated to play crucial and multifaceted roles in human health and disease. Aberrant NNMT expression has been observed in various diseases, including cancer [36; 37], metabolic disorders [38], and neurodegenerative diseases [39]. Consequently, NNMT inhibitors have emerged as promising therapeutic agents for these diseases. Competitive inhibitors that displace SAM or nicotinamide are based on NNMT substrate binding competition. However, it is evident that targeting only one substrate pocket may not be sufficient for effective NNMT inhibition. Therefore, dual-target NNMT inhibitors have been developed and can bind to both substrate-binding pockets, thereby enhancing inhibitor potency and selectivity. However, the cellular and in vivo activity of these inhibitors is limited due to poor cellular uptake. Current research on dual-target inhibitors focuses on optimizing the structural features based on the functional groups of the NAM and SAM substrate-binding pockets [40; 41; 42; 43; 44]. Additionally, covalently binding compounds, natural products, and macrocyclic peptides can also serve as alternative NNMT inhibitors [45; 46]. NMN can be synthesized by NRK from nicotinamide ribose, which is another substrate of the salvage pathway. [PERSON] et al. [47] utilized NR as a direct substrate and ATP as a co-substrate to efficiently produce NMN through the catalytic action of _S.