Efficient Synthesis of Purine Nucleoside Analogs by a New Trimeric Purine Nucleoside Phosphorylase from Aneurinibacillus migulanus AM007

Abstract

Purine nucleoside phosphorylases (PNPs) are promising biocatalysts for the synthesis of purine nucleoside analogs. Although a number of PNPs have been reported, the development of highly efficient enzymes for industrial applications is still in high demand. Herein, a new trimeric purine nucleoside phosphorylase (AmPNP) from Aneurinibacillus migulanus AM007 was cloned and heterologously expressed in Escherichia coli BL21(DE3). The AmPNP showed good thermostability and a broad range of pH stability. The enzyme was thermostable below 55 °C for 12 h (retaining nearly 100% of its initial activity), and retained nearly 100% of the initial activity in alkaline buffer systems (pH 7.0-9.0) at 60 °C for 2 h. Then, a one-pot, two-enzyme mode of transglycosylation reaction was successfully constructed by combining pyrimidine nucleoside phosphorylase (BbPyNP) derived from Brevibacillus borstelensis LK01 and AmPNP for the production of purine nucleoside analogs. Conversions of 2,6-diaminopurine ribonucleoside (1), 2-amino-6-chloropurine ribonucleoside (2), and 6-thioguanine ribonucleoside (3) synthesized still reached >90% on the higher concentrations of substrates (pentofuranosyl donor: purine base; 20:10 mM) with a low enzyme ratio of BbPyNP: AmPNP (2:20 μg/mL). Thus, the new trimeric AmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.

Keywords: enzyme ratio; purine nucleoside analogs; thermostability; trimeric nucleoside phosphorylase.

Figure 1

Phylogenetic analysis of purine nucleoside phosphorylases (PNPs). AhPNP (GenBank:WP_049045821) from Aeromonas hydrophila; BcPNP (GenBank:WP_098273438) from Bacillus cereus; BhPNP (GenBank: WP_010897696) from Bacillus halodurans; BtPNP (GenBank: AAX46392) from Bos taurus; Bs3PNP (GenBank: WP_015714207) from Bacillus subtilis; Bs6PNP (GenBank: WP_044428467) from Bacillus subtilis; DrPNP (GenBank: NP_998476) from Danio rerio; EcPNP (GenBank: AAN83888) from Escherichia coli; FhPNP (GenBank: XP_012736771) from Fundulus heteroclitus; GsPNP (GenBank: WP_053414649) from Geobacillus stearothermophilus; HaPNP (GenBank:KFP95377) from Haliaeetus albicilla; HsPNP (GenBank: NP_000261) from Homo sapiens; MmPNP (GenBank: NP_038660) from Mus musculus; SsPNP (GenBank: WP_009988635) from Sulfolobus solfataricus; StPNP (GenBank: WP_011681178) from Streptococcus thermophilus; TtPNP (GenBank: WP_096412123) from Thermus thermophilus; and XtPNP (GenBank: NP_001006720) from Xenopus tropicalis.

Figure 2

Molecular weight analysis of AmPNP. (a) Dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified AmPNP. Lane M: marker; Lane 1: crude enzymes; Lane 2: enzyme of AmPNP purified by Ni²⁺ column. (b) Analysis of AmPNP by gel filtration chromatography. The condition was conducted as following: an ÄKTA pure system and a column Superdex 200 Increase 10/300 GL were used. The standard proteins and the purified AmPNP sample were dissolved in the buffer (0.01 M phosphate buffer, 0.14 M NaCl, pH 8.0) with the final concentration of 2 mg/mL, respectively. For analysis, the flow rate was set at 0.3 mL/min and the inject volume was 500 μL.

Scheme 1

Phosphorylation of inosine with AmPNP.

Figure 3

Effects of temperature and pH on the activity and stability of AmPNP. (a) Effect of temperature on the activity of AmPNP. The highest activity was set to 100%. (b) Effects of temperature on stability of AmPNP. The initial activity was set to 100%. (c) Effect of pH on the activity determined by assaying the activity at 60 °C in 10 mM Na₂HPO₄/KH₂PO₄ buffer. The highest activity was set to 100%. (d) Effect of pH on stability determined by assaying the residual activity of AmPNP after 2 h at 60 °C and different pH values in 10 mM Na₂HPO₄/KH₂PO₄ buffer. The highest activity was set to 100%. The activity of AmPNP was determined in 10 mM Na₂HPO₄/KH₂PO₄ buffer (pH 8.0) with 10 mM inosine.

Scheme 2

Enzymatic synthesis of 2-amino-6-chloropurine ribonucleoside.

Figure 4

Effect of temperature and pH on biosynthesis of 2-amino-6-chloropurine ribonucleoside BbPyNP: AmPNP in a ratio of 2:20 μg/mL. The reaction mixture (1.0 mL) contained 20 mM uridine and 10 mM 2-amino-6-chloropurine. (a) The reaction was conducted in a thermostatic oscillator at a pH of 8.0 (20 mM Na₂HPO₄/KH₂PO₄) with temperature ranging from 30 °C to 65 °C for 36 h. (b) The reaction was carried out at a temperature of 55 °C for 60 h in 20 mM Na₂HPO₄/KH₂PO₄ buffer systems with pH ranging from 5.0 to 8.0.

Figure 5

Effect of the ratio of BbPyNP: AmPNP on biosynthesis of 2-amino-6-chloropurine ribonucleoside. Process for synthesis of 2-amino-6-chloropurine ribonucleoside was shown in Scheme 2. Using 20 mM uridine and 10mM 2-amino-6-chloropurine, the reaction mixture (1.0 mL) contained purified BbPyNP (85 IU/mg for uridine) and purified AmPNP (83 IU/mg) at different ratios (the concentration of AmPNP was set at 20 μg/mL). The reaction was carried out at pH 7.0 (20 mM Na₂HPO₄/KH₂PO₄) and a temperature of 55 °C for 36 h, followed by analysis.