Structure‐Guided Tuning of a Selectivity Switch towards Ribonucleosides in Trypanosoma brucei Purine Nucleoside 2′‐Deoxyribosyltransferase

Abstract

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.

N-Ribosyltransferase From Archaeoglobus veneficus: A Novel Halotolerant and Thermostable Biocatalyst for the Synthesis of Purine Ribonucleoside Analogs

“…These results agree with the hypothesis derived from the bioinformatic analysis (Figures 1, 2B) suggesting a strict preference of AvNRT for ribonucleosides. Furthermore, they are also in line with those previously reported for TbPDT demonstrating that the Tyr5→Phe replacement enhances the ability of this enzyme to accept ribonucleosides as substrates (Del Arco et al, 2019b).…”

“…The RCD+ server (López-Blanco et al, 2016) was employed to generate alternative loop conformations of the intrinsically disordered regions. The AMBER force field (Salomon-Ferrer et al, 2013) was used for progressive energy refinement in explicit solvent followed by molecular dynamics and simulated annealing according to a previously described protocol (Del Arco et al, 2019b). PyMOL was used for structure visualization, molecular editing, and figure preparation (Delano, 2002).…”

“…However, neither NMP nor dNMP glycosidases have been reported to be active in transglycosylation reactions to date. The different specificity between NMP and dNMP glycosidases is thought to be mostly due to the presence of either a Phe or a Tyr side chain close to the catalytic Glu residue (Sikowitz et al, 2013;Del Arco et al, 2019b) (Figure 1).…”

“…Despite their great potential for performing important chemical transformations, the use of NDTs as industrial biocatalysts for the synthesis of nucleoside analogs is often hindered by their strict preference for 2 ′ -deoxyribonucleosides. In this respect, several attempts have been made for tailoring NDT active sites toward new substrate specificities on the ribose moiety (Kaminski et al, 2008;Kaminski and Labesse, 2013;Del Arco et al, 2019b;Li et al, 2019). Additionally, most of the reported NDTs up to date are mesophilic enzymes, with the exception of NDTs from cold-adapted Bacillus psychrosaccharolyticus (Fresco-Taboada et al, 2014) and the extremophilic cyanobacteria Chroococcidiopsis thermalis PCC 7203 (CtNDT) (Del Arco et al, 2018a).…”

Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates

“…In this sense, the use of enzymes from purine and pyrimidine salvage pathway as biocatalysts for the synthesis of nucleosides and nucleotides has been extensively reported ( Mikhailopulo, 2007 ; Fernández-Lucas et al, 2010 ; Fresco-Taboada et al, 2013; Fernández-Lucas , 2015 ; Lapponi et al, 2016 ; Del Arco and Fernández-Lucas, 2017; Ding and Ou , 2017 ; Lewkowicz and Iribarren, 2017 ; Serra et al, 2017 ; Acosta et al, 2018 ; Del Arco et al, 2018b , 2019a ; Pérez et al, 2018 ; Kamel et al, 2019 ; Ubiali and Speranza, 2019 ).…”