Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases fromThermus thermophilusHB8

Abstract

Purine phosphoribosyltransferases, purine PRTs, are essential enzymes in the purine salvage pathway of living organisms. They are involved in the formation of C-N glycosidic bonds in purine nucleosides-5 0 -monophosphate (NMPs) through the transfer of the 5-phosphoribosyl group from 5-phospho-a-D-ribosyl-1-pyrophosphate (PRPP) to purine nucleobases in the presence of Mg 2þ . Herein, we report a simple and thermostable process for the one-pot, one-step synthesis of some purine NMPs using xanthine phosphoribosyltransferase, XPRT or adenine phosphoribosyltransferase, APRT2, from Thermus thermophilus HB8. In this sense, the cloning, expression and purification of TtXPRT and TtAPRT2 is described for the first time. Both genes, xprt and aprt2 were expressed as his-tagged enzymes in E. coli BL21(DE3) and purified by a heat-shock treatment, followed by Ni-affinity chromatography and a final, polishing gel-filtration chromatography. Biochemical characterization revealed TtXPRT as a tetramer and TtAPRT2 as a dimer. In addition, both enzymes displayed a strong temperature dependence (relative activity >75% in a temperature range from 70 to 90 C), but they also showed very different behaviour under the influence of pH. While TtXPRT is active in a pH range from 5 to 7, TtAPRT2 has a high dependence of alkaline conditions, showing highest activity values in a pH range from 8 to 10. Finally, substrate specificity studies were performed in order to explore their potential as industrial biocatalyst for NMPs synthesis. ARTICLE HISTORY

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

“…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.…”

“…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.…”

“…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.…”

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

“…In this context, the enzymatic synthesis of NMPs shows many advantages, such as one‐pot reactions under mild conditions, high stereo‐ and regioselectivity, and an environmentally friendly technology. Therefore, many different enzymes have been employed as valuable catalysts for mono‐ or multienzymatic synthesis of NMPs, including nucleoside kinases (NK), acid phosphotransferases (AP/PTase), 5′‐phosphodiesterases, and phosphoribosyltransferases (PRT) …”