Reshaping the binding pocket of purine nucleoside phosphorylase for improved production of 2-halogenated-2′-deoxyadenosines†
Abstract
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.