Gaofei Liu,Tiantong Cheng,Jianlin Chu,Sui Li,Bingfang He
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) fromAneurinibacillus migulanusAM007 was cloned and heterologously expressed inEscherichia coliBL21(DE3). TheAmPNP 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 fromBrevibacillus borstelensisLK01 andAmPNP 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 ofBbPyNP:AmPNP (2:20 μg/mL). Thus, the new trimericAmPNP is a promising biocatalyst for industrial production of purine nucleoside analogs.Keywords:trimeric nucleoside phosphorylase;thermostability;purine nucleoside analogs;enzyme ratio
Gaofei Liu,Xin Tong,Jialing Wang,Bin Wu,Jianlin Chu,Yongchan Jian,Bingfang He
2-Halogenated-2′-deoxyadenosines are important antiviral and anticancer drugs. However, natural enzymes display low activities in the biosynthesis of 2-halogenated-2′-deoxyadenosines, and the results are unsatisfactory because of the steric impediment created by the halogenated substituent group in purine. In this study, a semi-rational design was performed to enhance the activity of purine nucleoside phosphorylase from Aneurinibacillus migulanus AM007 (AmPNP) for the biosynthesis of 2-halogenated-2′-deoxyadenosines. Small and smart libraries of AmPNP were generated by stepwise site-directed evolution using iterative combinatorial mutations. The best AmPNP mutant, M3 (N233D/E191Q/Y190V/M249I), exhibited a satisfactory phosphorolysis activity towards 2′-deoxyadenosine, with a 144.75-fold improvement compared with that of wild-type AmPNP. In a coupled reaction of the M3 mutant and Brevibacillus borstelensis LK01 pyrimidine nucleoside phosphorylase, the conversion rates remarkably improved and reached 89.94% (cladribine) and 81.24% (2-fluoro-2′-deoxyadenosine) within 10 h, respectively. The substrate-binding cavity of the M3 mutant was reshaped to provide good accessibility for halogenated molecules, which led to a higher activity in the synthesis of 2-halogenated nucleosides. This directed evolution towards overcoming challenges in the biosynthesis of halogenated nucleosides may result in a remarkable economic impact on biotechnological nucleoside production.
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