## 3 Importance of ATP-Regeneration Systems in the Synthesis of 5\({}^{\prime}\)-NTPs  
A key parameter for cofactor-dependent enzymes is the total turnover number (TTN), which indicates the moles of product generated per mole of cofactor over its entire lifespan, serving as a measure of catalytic efficiency. In the absence of a regeneration system for group-transfer-cofactors like ATP, these cofactors must be used in stoichiometric amounts, making the process inefficient for industrial applications. However, by implementing a regeneration system, the TTN can be increased to values of \(\geq\)10\({}^{5}\)[5]. A recent analysis highlighted the efficiency of ATP regeneration systems across various reactions [105]. Various ATP regeneration systems, such as PK/phosphoenolpyruvate, Ack/acetyl phosphate, and PPK/polyphosphate, are frequently employed. While PK/phosphoenolpyruvate and Ack/acetyl phosphate have been used for decades, the PPK/polyphosphate system is gaining increasing attention for its cost-effectiveness only recently [59; 79; 92; 97]. Although _Escherichia coli_ PPK was first mentioned for ATP regeneration in the 1950s [74], it was not until several decades later that interest in its role in metabolism and nucleotide synthesis grew significantly. Each ATP regeneration system has distinct advantages and drawbacks depending on the enzyme and phosphate donor used. Acetyl phosphate is an affordable and widely used phosphate donor. It can be easily synthesized from inexpensive materials and used as a crude solution in some applications [42]. However, it is less stable than alternatives [106]. To address this issue, a pyruvate oxidase-based synthesis of acetyl phosphate was developed. To overcome the limitations of this method, including hydrogen peroxide formation and  
Figure 8: Transglycosylation reactions catalyzed by an engineered NdT and using 5\({}^{\prime}\)-dNTTPs as substrate. 5-BrU: 5-bromo uracil, 5-BrdUTP: 5-bromo dUTP, 5-FU: 5-fluoro uracil, 5-F-dUTP: 5-fluoro dUTP, 2,6-D: 2,6-diamino purine, 2,6-dTPTP: 2,6-diamino purine 5\({}^{\prime}\)-triphosphate, 6-Cl-P: 6-chloro purine, 6-Cl-dTPTP: 6-chloro purine 5\({}^{\prime}\)-triphosphate, 2,6-Cl-P: 2,6-dichloropurine, 2,6-Cl-dTPTP: 2,6-dichloropurine 5\({}^{\prime}\)-triphosphate. poor oxygen mass transfer from the gas to the liquid phase, an electrochemical oxidation of FADH\({}_{2}\), the cofactor of pyruvate oxidase, has recently been proposed [107]. Another drawback of AcKs is their sensitivity to oxidation, which necessitates chemical protection to prevent inactivation [106]. In contrast, phosphoenolpyruvate is highly stable and thermodynamically the most efficient phosphate donor due to its high free enthalpy of phosphate hydrolysis, which results in nearly irreversible phosphate transfer to an acceptor [5; 106]. However, its industrial use is limited by its relatively high cost and a potential kinase inhibition by pyruvate at concentrations around 10 mM [106]. Polyphosphate has become increasingly popular as a secondary phosphate donor due to its low cost and stability across a broad pH range, though it has some limitations [70]. Many commercial polyphosphate products consist of mixtures with varying chain lengths [108], which can complicate studies that require a specific chain length, particularly if the enzyme used has a preference. Additionally, polyphosphate and free phosphate can precipitate with magnesium or other essential cations, potentially affecting enzyme activity. Furthermore, polyphosphates' lower free enthalpy values compared to ATP result in lower equilibrium constants, requiring a large excess of polyphosphate for efficient reactions [70]. Another ATP regeneration system, using CK and phosphocreatine, has also been explored. Phosphocreatine, like acetyl phosphate, is inexpensive but unstable [106]. CK has a low K\({}_{\text{m}}\) for ADP, which is advantageous for certain applications [70]. Phosphate donor regeneration systems are incorporated into enzymatic reactions for several reasons.