### Enzymes Involved in Nucleotide Metabolism
#### 1.2.1 Enzymes Involved in the Synthesis of Natural and Modified 5'-NMPs  
5'-NMPs can be synthesized from nucleobases or nucleosides, or they can be derived from the enzymatic degradation of RNA and DNA, such as by nucleases [24] (Figure 2). Purine and pyrimidine phosphoribosyltransferases (PRTs), like adenine PRT, hypoxanthine-guanine PRT, uracil PRT, and orotate PRT, catalyze the coupling of a nucleobase with PRPP to form the respective 5'-NMP (Figure 1). The classification of PRTs and their use in the synthesis of various natural and base-modified NMPs was reviewed by [PERSON] and [PERSON] in 2017 [25]. Nucleosides featuring a C-glycosic bond instead of the typical \(N\)-glycosic bond, such as pseudouridine-5'-monophosphate, are formed by pseudouridylate synthase (PUS or YeiN). This enzyme couples the nucleobase directly to ribose-5-phosphate (PR) [26] (Figure 1). The authors demonstrated that this enzyme can be applied on a milligram scale to produce a variety of sugar- and base-modified pseudouridine-5'-monophosphate analogs. Alternatively, 5'-NMPs are generated by the 5'-monophosphorylation of nucleosides through various enzymes and phosphate donors. Nucleoside kinases (NKs) and deoxynucleoside kinases (dNKs) are the most well-known enzymes that catalyze the nearly irreversible 5'-monophosphorylation of (deoxy)nucleosides using 5'-(d)NTPs as phosphate donors (Figure 1). General dNKs accept a broad range of nucleoside substrates, while variants such as adenosine kinase or uridine-cytidine kinase have a narrower substrate spectrum. Due to their crucial role in activating antiviral and anticancer nucleoside drugs in the human body, both human and viral (d)NKs have been extensively studied [22]. Given that most antiviral and anticancer nucleoside drugs are deoxynucleoside analogs, much of the research has focused on dNKs rather than NKs. The broad substrate specificity of human (d)NKs is evident in the variety of nucleoside analog drugs that are activated to 5'-NTPs. These analogs include open-ring sugars like ganciclovir, open-ring bases such as ribavirin and minoribine, and nucleosides with fluorine, azide, or no functional groups at the 2' and 3'-positions, such as gemcitabine, zidovudine, and stavudine, as well as those with unusual sugar conformations like cytarabine, arabinosyl guanine, and lamivudine. Other examples include large base modifications like Brivudine; halogenated derivatives like cladribute, fludarabine, or 5-fluorouracil; thiolated bases  
Figure 2: Synthesis of 5′-NTPs from RNA. The figure was adapted from [PERSON] and [PERSON] (1990) [24]. ADK: adenosine kinase, Ack: acetate kinase, AcP: acetyl phosphate. like 2-thiocytidine and 4-thiouridine; and carbocyclic derivatives such as aristeromycin, cyclopentenyl cytosine, or carbocyclic deoxyguanosine [22; 27; 28; 29]. However, not only mammalian cells but also other organisms harbor wide-spectrum enzymes. _Methanoaldococcus jannaschii_ and _Thermoplasma acidophilum_ possess wide-spectrum NKs that exhibit activity toward inosine, cytidine, guanosine, and adenosine [30; 31]. Several insects, including the fruit fly _Drosophila melanogaster_ (_Dmd_NK), silk moths, and mosquitoes, have multisubstrate dNKs [32]. _Dmd_NK, with its remarkable substrate spectrum and activity, has been the focus of extensive research [23; 33; 34; 35] and has been applied in the synthesis of various NMP analogs [36]. Additionally, viral dNKs, such as the thymidine kinase of _Herpes simplex_ virus (HSV), exhibit broad substrate specificity, making them another important area of study. In addition to (d)NKs, several other enzymes are involved in the phosphorylation of nucleosides and their analogs, including 5\({}^{\prime}\)-nucleotidases [37; 38], nonspecific acid phosphatases [39; 40] or nucleoside phosphotransferases [41], and even protein kinases. It is worth highlighting nonspecific acid phosphatases here.