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.
Highlights
Up to now, nucleoside analogs have been significant sources of antiviral and anticancer drugs [1,2,3], among which purine nucleoside analogs are widely used for the treatment of leukemia [4,5] and are important precursors of several drugs [6,7]
The amino acid sequence of a new trimeric purine nucleoside phosphorylase (AmPNP) from A. migulanus AM007 was analyzed with sequence alignment (Supplementary Material, Table S1), which had a high identity of 66.1% with trimeric BsPNP
2-Chloro-20 -deoxyadenosine (Cladribine) was formed at a yield of 60.4%, and an antiviral drug ribavirin was formed with a yield of 73.7% with the transglycosylation reaction. These results indicate that AmPNP is an excellent biocatalyst for the industrial production of purine nucleoside analogs
Summary
Nucleoside analogs have been significant sources of antiviral and anticancer drugs [1,2,3], among which purine nucleoside analogs are widely used for the treatment of leukemia [4,5] and are important precursors of several drugs [6,7]. For the effective formation of purine nucleosides, PyNPs and PNPs are usually combined in a one-pot, two-enzyme reaction system. The hexametric PNPs are mostly isolated from bacterial species like Salmonella typhimurium [19] and Aeromonas hydrophila [20], which exhibit a phosphorolysis activity against adenosine and some derivatives, and have a subunit molecular mass of approximately 26 kDa. trimeric PNPs have good thermal stability, they have narrower substrate specificity. Zhou et al used GtPNP and TtPyNP (enzyme ratio; 2:1) to synthesize purine nucleoside analogs with high yield [16]. Efficient synthesis of purine nucleosides analogs was successfully achieved in the one-pot, two-enzyme transglycosylation reaction catalyzed by AmPNP and PyNP derived from. The ratio of BbPyNP and AmPNP was optimized for the one-pot, two-enzyme reaction, greatly reducing the production cost
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