Thermus thermophilus Nucleoside Phosphorylases Active in the Synthesis of Nucleoside Analogues

Abstract

b Cells extracts from Thermus thermophilus HB27 express phosphorolytic activities on purines and pyrimidine nucleosides. Five putative encoding genes were cloned and expressed in Escherichia coli, and the corresponding recombinant proteins were purified and studied. Two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides in vitro, and the three were named TtPNPI, TtPNPII, and TtPyNP, respectively. The optimal temperature for the activity of the three enzymes was beyond the water boiling point and could not be measured accurately, whereas all of them exhibited a wide plateau of optimal pHs that ranged from 5.0 to 7.0. Analytical ultracentrifugation experiments revealed that TtPNPI was a homohexamer, TtPNPII was a monomer, and TtPyNP was a homodimer. Kinetic constants were determined for the phosphorolysis of the natural substrates of each enzyme. Reaction tests with nucleoside analogues revealed critical positions in the nucleoside for its recognition. Activities with synthetic nucleobase analogues, such as 5-iodouracil or 2,6-diaminopurine, and arabinosides were detected, supporting that these enzymes could be applied for the synthesis of new nucleoside analogs with pharmacological activities.

Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides

“…5–8 times higher in the cases of DgPNP and GtPNP. Similar remarkable differences in the substrate affinity were also described for PNPs from T. thermophilus and PfMTAP . The k cat values of the nucleoside phosphorylases assessed for the natural substrates dIno and dAdo point to a very high catalytic activity of GtPNP (cf.…”

“…The aforementioned results of this study and data from previous studies [17] prompted us to investigate the synthesis of adenine 2′-deoxyfluoro-ribo-and -arabino-nucleosides, dAdo 2′F (17) and dAdo 2′F (18), respectively, using adenine as an acceptor and the respective 2′-fluorinated uracil nucleosides, dUrd 2′F (13) and dUrd 2′F (14), as donors of the pentofuranose residues (Scheme 3). Preliminary experiments showed the formation of: (a) dAdo 2′F using TtPyNP and DgPNP as biocatalysts (55°C, 24 h) with a yield of 23%, and (b) dAdo 2′F employing a TtPyNP and ApM-TAP combination of enzymes (80°C, 24 h) with a yield of 24%.…”

Revisiting the methionine salvage pathway and its paralogues

“…The lesson from these and related works is that there is a considerable range of nucleoside phosphorolysis activities that could be used as a rich mine for developing unique processes of metabolic engineering, or for uncovering novel drug targets. Nucleoside phosphorylases from E. coli or B. subtilis have previously been used for the synthesis of pharmacologically active compounds, but many more organisms may be used in a variety of conditions to develop microbial synthesis of nucleoside analogues (Almendros et al, 2012). Another lesson is that we should not forget that survey of old work may be of considerable value for preparing the future.…”

Immobilization of thermostable nucleoside phosphorylases on MagReSyn® epoxide microspheres and their application for the synthesis of 2,6-dihalogenated purine nucleosides

“…Thermostable NPs from thermophilic microorganisms, retaining the high regio-and stereo-selectivity of the mesophilic NPs, have further advantages that are not only associated with an increased thermostability but also with a broader substrate spectrum and a wider tolerance to many stresses [3][4][5][6][7]. Among them, purine nucleoside phosphorylase (PNP, EC 2.4.2.1) from Geobacillus thermoglucosidasius (GtPNP) [3] and pyrimidine nucleoside phosphorylase (PyNP, EC 2.4.2.2) from Thermus thermophiles (TtPyNP) [4] have shown such promising properties.…”