Enzymatic Production of Non‐Natural Nucleoside‐5′‐Monophosphates by a Thermostable Uracil Phosphoribosyltransferase

Abstract

The use of enzymes as biocatalysts applied to synthesis of modified nucleoside‐5′‐monophosphates (NMPs) is an interesting alternative to traditional multistep chemical methods which offers several advantages, such as stereo‐, regio‐, and enantioselectivity, simple downstream processing, and mild reaction conditions. Herein we report the recombinant expression, production, and purification of uracil phosphoribosyltransferase from Thermus themophilus HB8 (TtUPRT). The structure of TtUPRT has been determined by protein crystallography, and its substrate specificity and biochemical characteristics have been analyzed, providing new structural insights into the substrate‐binding mode. Biochemical characterization of the recombinant protein indicates that the enzyme is a homotetramer, with activity and stability across a broad range of temperatures (50–80 °C), pH (5.5–9) and ionic strength (0–500 mm NaCl). Surprisingly, TtUPRT is able to recognize several 5 and 6‐substituted pyrimidines as substrates. These experimental results suggest TtUPRT could be a valuable biocatalyst for the synthesis of modified NMPs.

One-Pot Multi-Enzymatic Production of Purine Derivatives with Application in Pharmaceutical and Food Industry

“…Modified nucleosides have been traditionally synthesized by different chemical methods, requiring the use of protection-deprotection steps, organic solvents and chemical reagents [4][5][6][7][8][9][10][11]. In this sense, LdNDT has shown to be an interesting sustainable alternative to traditional chemical methods for the synthesis of many different purine nucleosides.…”

“…Covalent immobilization of proteins usually occurs via multipoint attachment through the region with higher density of primary amino groups, especially with ε-NH 2 from lysine residues [22]. Due to this, covalent immobilization techniques usually employ long reaction times (2-24 h) and alkaline conditions (pH [8][9][10]. In this way, the exceptional stability that is displayed by LdNDT and TtHGXPRT [11] under alkaline conditions suggests that they could be efficiently immobilized.…”

“…In this context, the enzymatic synthesis of NAs and NMPs shows many advantages, such as one-pot reactions under mild conditions, high stereo-, and regioselectivity, and an environmentally friendly technology [4][5][6][7][8][9][10][11].…”

“…On the contrary, the salvage pathway is composed by a group of reutilization routes by which the cell can satisfy its purine requirements from endogenous and/or exogenous sources of preformed purines. In this regard, numerous enzymes from purine salvage pathway have become valuable catalysts for mono or multi-enzymatic synthesis of nucleosides and nucleotides, such as nucleoside kinases (NKs) [12][13][14][15], phosphoribosyltransferases [7,[9][10][11], nucleoside phosphorylases [4,8,16,17], 2′-deoxyribosyltransferases [5,[18][19][20], among others.…”

Purine and pyrimidine salvage pathway in thermophiles: a valuable source of biocatalysts for the industrial production of nucleic acid derivatives

“…Several thermophilic UPRTs have been described, such as dimeric UPRT from Bacillus caldolyticus, tetrameric UPRTs from S. solfutaricus, Aquifex aeolicus, and T. thermophilus (Del Arco et al 2017a). Among them, we wish to highlight the thermal activity and stability of UPRTs from B. caldolyticus (relative activity ≈ 60% at 70°C for 10 min), Sulfolobus shibatae (negligible loss of activity at 80°C for 1 h), and T. thermophilus (negligible loss of activity at 70°C for 4 h and a half-life over 70 min at 80°C) (Del Arco et al 2017a).…”