Issue 13, 2021

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.

Graphical abstract: Reshaping the binding pocket of purine nucleoside phosphorylase for improved production of 2-halogenated-2′-deoxyadenosines

Supplementary files

Article information

Article type
Paper
Submitted
19 Dec 2020
Accepted
02 May 2021
First published
20 May 2021

Catal. Sci. Technol., 2021,11, 4439-4446

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

G. Liu, X. Tong, J. Wang, B. Wu, J. Chu, Y. Jian and B. He, Catal. Sci. Technol., 2021, 11, 4439 DOI: 10.1039/D0CY02424D

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements