Thermus thermophilus nucleoside phosphorylases active in the synthesis of nucleoside analogues

Abstract

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.

Fig 1

Scheme of the reaction catalyzed by nucleoside phosphorylases. (A) Phosphorolytic and the synthetic pathways. (B) Synthesis of nucleosides by coupled NP activity reactions. A donor nucleoside is cleaved by an NP, yielding a free nucleobase (B₁) and a pentose-1-P. The pentose is then used in a new reaction by either the same or a different NP, along with an acceptor nucleobase (B₂) to synthesize a new nucleoside.

Fig 2

Purification of proteins TTC0188, TTC1491, TTC0194, TTC1070, and TTC1412. The indicated proteins were overproduced from the corresponding pET22b derivatives in E. coli BL21 cells. Lanes: 1, whole extract obtained after cell disruption by sonication; 2, soluble fraction after incubation of the whole extract at 80°C for 1 h; 3, purification by ion-exchange chromatography of the heat-treated extract; M, size markers (in kilodaltons).

Fig 3

Determination of T. thermophilus HB27 NP oligomeric state by analytical ultracentrifugation. (A) TtPNPI, 0.35 mg/ml in 50 mM sodium phosphate (pH 7.0) at 20°C, 50,000 × g. (B) TtPNPII, 0.22 mg/ml in 50 mM sodium phosphate (pH 7.0) at 20°C, 50,000 × g. (C) TtPyNP, 0.53 mg/ml in 50 mM sodium phosphate (pH 7.0) at 20°C, 50,000 × g.

Fig 4

Optimization of reaction conditions, i.e., the phosphate buffer concentration (A), medium pH (B), and temperature (C) in phosphorolytic reactions carried out by TtPNPI (■), TtPNPII (▲), and TtPyNP (●). The values on the y axis correspond to the residual activity, where 1 is the activity under standard conditions (50 mM sodium phosphate buffer [pH 7.0], 65°C).