### Enzymes Involved in Nucleotide Metabolism
#### 1.2.3 Enzymatic 5'-NTP Synthesis  
The primary route for 5\({}^{\prime}\)-(d)NTP synthesis involves the phosphorylation of 5\({}^{\prime}\)-(d)NDPs by NDP kinases (NDPKs), which utilize other 5\({}^{\prime}\)-(d)NTPs as phosphate donors (Figure 1). NDPKs demonstrate low specificity for both the phosphate acceptor and donor. A wide range of nucleobases are accepted, as the binding residues form a cleft with aromatic stacking, rather than making specific polar interactions with surrounding amino acids [54; 55]. There are minimal restrictions on the 2\({}^{\prime}\)-position of the sugar moiety, allowing both ribose and deoxyribose nucleotides to be converted. However, the presence of the 3\({}^{\prime}\)-hydroxyl group is essential for efficient performance [54]. Derivatives with modifications on the phosphate groups, such as thio-phosphates [56] or sulfuryl-groups [57], have been shown to be weak substrates. A wide variety of modified 5\({}^{\prime}\)-NTPs have been produced using NDPKs so far, including 8-hydroxy-dGTP, 2-hydroxy-dATP [58], five azide carboxamide 5\({}^{\prime}\)-dNTPs [59], and pyrimidine and purine 5\({}^{\prime}\)-NTPs such as arabinosyl CTP, 2\({}^{\prime}\)-deoxy-2\({}^{\prime}\)-azido-CTP and -UTP, 2\({}^{\prime}\)3\({}^{\prime}\)-dideoxy-3\({}^{\prime}\)-azido-dATP, and 2\({}^{\prime}\)-deoxy-2\({}^{\prime}\)-amino-GTP [60]. Other widely used enzymes for 5\({}^{\prime}\)-NTP synthesis include pyruvate kinase (PK), acetate kinase (AcK), and creatine kinase (CK), which are also commonly used in in vitro (d)ATP regeneration systems. These enzymes show a broad substrate spectrum and have been employed to synthesize a variety of 5\({}^{\prime}\)-NTPs and their analogs. PK, using phosphophorus are the phosphate donor, was able to generate all natural 5\({}^{\prime}\)-(d)NTPs from the respective 5\({}^{\prime}\)-(d)NDPs [61]. Additionally, it was used for synthesizing ribavirin-5\({}^{\prime}\)-TP [62], arabinosyl CTP [63], and 8-azaguanosine-5\({}^{\prime}\)-triphosphate [64]. CK, using phosphoborene-time as phosphate donor, was applied for the synthesis of 8-L-2\({}^{\prime}\),3\({}^{\prime}\)-dideoxy-ATP [65] and 2-fluoro-ATP [66]. When _Escherichia coli_ cells were found to remain viable even with disrupted NDPK and PK genes, it was also discovered that NMPK exhibited NDPK activity [67]. Further research demonstrated NDPK activity in human UMP-CMP kinase and adenylate kinase 9, which are capable of converting 5\({}^{\prime}\)-(d)NMPs sequentially to the corresponding 5\({}^{\prime}\)-(d)NTPs [68]. However, this capability has not yet been utilized in 5\({}^{\prime}\)-NTP production processes. Interconversions between 5\({}^{\prime}\)NTPs are also possible, as seen in the UTP to CTP conversion catalyzed by CTP synthetase (Figure 1). Additionally, some nucleoside-specific deaminases have demonstrated minor activity toward 5\({}^{\prime}\)-NTPs [69]. 2.4 Conversion of 5\({}^{\prime}\)-NMPs and 5\({}^{\prime}\)-NDPs to 5\({}^{\prime}\)-NTPs by Polyphosphate kinases  
Polyphosphate kinases (PPKs) catalyze the conversion of 5\({}^{\prime}\)-NMPs and 5\({}^{\prime}\)-NDPs into 5\({}^{\prime}\)-NTPs, utilizing polyphosphate as a cost-effective source of inorganic phosphate (Figure 1). This ability makes PPKs valuable enzymes for ATP regeneration starting from AMP or ADP. Recent reviews have highlighted the use of PPK enzymes for 5\({}^{\prime}\)-NTP and 5\({}^{\prime}\)-NDP regeneration in coupled enzymatic reactions [70; 71]. In 2014, a classification of PPKs was proposed based on phylogenetic and functional analyses, categorizing these enzymes into two distinct families with no sequence similarity [72]. PPK1 enzymes generally favor polyphosphate synthesis, while PPK2 enzymes are inclined toward polyphosphate degradation.