Structure-Guided Tuning of a Selectivity Switch towards Ribonucleosides in Trypanosoma brucei Purine Nucleoside 2'-Deoxyribosyltransferase.,ChemBioChem

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Structure-Guided Tuning of a Selectivity Switch towards Ribonucleosides in Trypanosoma brucei Purine Nucleoside 2'-Deoxyribosyltransferase.
ChemBioChem ( IF 2.6 ) Pub Date : 2019-10-18 , DOI: 10.1002/cbic.201900397
Jon Del Arco ₁ , Alberto Mills ₂ , Federico Gago ₂ , Jesús Fernández-Lucas _{1,

3}

Affiliation

The use of nucleoside 2'-deoxyribosyltransferases (NDTs) as biocatalysts for the industrial synthesis of nucleoside analogues is often hindered by their strict preference for 2'-deoxyribonucleosides. It is shown herein that a highly versatile purine NDT from Trypanosoma brucei (TbPDT) can also accept ribonucleosides as substrates; this is most likely because of the distinct role played by Asn53 at a position that is usually occupied by Asp in other NDTs. Moreover, this unusual activity was improved about threefold by introducing a single amino acid replacement at position 5, following a structure-guided approach. Biophysical and biochemical characterization revealed that the TbPDTY5F variant is a homodimer that displays maximum activity at 50 °C and pH 6.5 and shows a remarkably high melting temperature of 69 °C. Substrate specificity studies demonstrate that 6-oxopurine ribonucleosides are the best donors (inosine>guanosine≫adenosine), whereas no significant preferences exist between 6-aminopurines and 6-oxopurines as base acceptors. In contrast, no transferase activity could be detected on xanthine and 7-deazapurines. TbPDTY5F was successfully employed in the synthesis of a wide range of modified ribonucleosides containing different purine analogues.

中文翻译：

在布氏锥虫嘌呤嘌呤核苷2'-脱氧核糖基转移酶中向核糖核苷的选择性转换的结构导向的调节。

核苷2'-脱氧核糖基转移酶（NDTs）用作核苷类似物的工业合成的生物催化剂经常受到严格偏爱2'-脱氧核糖核苷的阻碍。本文显示，来自布鲁氏锥虫（TbPDT）的高度通用的嘌呤NDT也可以接受核糖核苷作为底物；这很可能是由于Asn53在其他NDT中通常被Asp占据的位置所起的独特作用。此外，按照结构指导的方法，通过在位置5引入单个氨基酸替代，将这种异常活性提高了约三倍。生物物理和生化特征表明，TbPDTY5F变体是同型二聚体，在50°C和pH 6.5下显示最大活性，并显示69°C的极高熔化温度。底物特异性研究表明6-氧嘌呤核糖核苷是最好的供体（肌苷>鸟苷≫腺苷），而6-氨基嘌呤和6-氧嘌呤作为碱基受体没有明显的偏爱。相反，在黄嘌呤和7-脱氮嘌呤上未检测到转移酶活性。TbPDTY5F已成功用于合成包含不同嘌呤类似物的多种修饰的核糖核苷。

更新日期：2019-10-19

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