Identification of a 2′-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries

Agota Aučynaitė,Rolandas Meškys,Daiva Tauraitė,Jaunius Urbonavičius,Rasa Rutkienė

doi:10.3390/molecules23112904

Agota Aučynaitė, Rolandas Meškys + Show 3 more

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https://doi.org/10.3390/molecules23112904

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Abstract

Ribose methylation is among the most ubiquitous modifications found in RNA. 2′-O-methyluridine is found in rRNA, snRNA, snoRNA and tRNA of Archaea, Bacteria, and Eukaryota. Moreover, 2′-O-methylribonucleosides are promising starting materials for the production of nucleic acid-based drugs. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding the metabolic fate and enzymes involved in the metabolism of 2′-O-alkyl nucleosides. The presented work focuses on the cellular degradation of 2′-O-methyluridine. A novel enzyme was found using a screening strategy that employs Escherichia coli uracil auxotroph and the metagenomic libraries. A 2′-O-methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA)—forming a part of a probable gene cluster that is involved in the degradation of 2′-O-methylated nucleosides. The RK9NH is functional in E. coli uracil auxotroph and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2′-O-methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts 5-fluorouridine, 5-fluoro-2′-deoxyuridine and 5-fluoro-2′-O-methyluridine into 5-fluorouracil, which suggests it could be employed in cancer therapy.

Highlights

Natural modified nucleotides are present in various kinds of nucleic acids and are most diverse in tRNA [1]
We hypothesized that an E. coli strain lacking the UMP de novo synthesis pathway would grow in the synthetic minimal media, if 20 -O-methyluridine would be converted into uracil (Figure 1) and subsequently used in the UMP salvage pathway
In order to search for genes supporting the growth of DH10B∆pyr cells on M9 minimal medium supplemented with 20 -O-methyluridine, several metagenomic libraries were transformed into these cells

Summary

Introduction

Natural modified nucleotides are present in various kinds of nucleic acids and are most diverse in tRNA [1]. Functions of the nucleotide modifications span from providing structural stability and increasing resistance to physiological degradation of nucleic acids to transcriptional regulation and even implications in the regulatory pathways of the cell [2,3,4,5,6,7,8,9,10,11]. The studies of modified nucleic acid degradation are limited. The biodegradation of modified nucleotides is understood mainly to the point of formation of nucleosides, even their subsequent conversion into heterocyclic bases is seldom described, with an exception of pseudouridine [21].

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