A novel nucleoside hydrolase from Lactobacillus buchneri LBK78 catalyzing hydrolysis of 2′-O-methylribonucleosides

Abstract

2'-O-Methylribonucleosides (2'-OMe-NRs) are promising raw materials for nucleic acid drugs because of their high thermal stability and nuclease tolerance. In the course of microbial screening for metabolic activity toward 2'-OMe-NRs, Lactobacillus buchneri LBK78 was found to decompose 2'-O-methyluridine (2'-OMe-UR). The enzyme responsible was partially purified from L. buchneri LBK78 cells by a four-step purification procedure, and identified as a novel nucleoside hydrolase. This enzyme, LbNH, belongs to the nucleoside hydrolase superfamily, and formed a homotetrameric structure composed of subunits with a molecular mass around 34 kDa. LbNH hydrolyzed 2'-OMe-UR to 2'-O-methylribose and uracil, and the kinetic constants were Km of 0.040 mM, kcat of 0.49 s(-1), and kcat/Km of 12 mM(-1) s(-1). In a substrate specificity analysis, LbNH preferred ribonucleosides and 2'-OMe-NRs as its hydrolytic substrates, but reacted weakly with 2'-deoxyribonucleosides. In a phylogenetic analysis, LbNH showed a close relationship with purine-specific nucleoside hydrolases from trypanosomes.

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

“…Multiple amino acid sequence alignment of RK9NH nucleoside hydrolase with RihB [ 33 , 39 ], RihA [ 33 ], RihC [ 33 ], and 2′- O -methylribonucleoside-specific nucleoside hydrolase from Lactobacillus buchneri LBK78 [ 27 ] ( Figure 3 ) reveals that the amino acid residues that form the active site of RihB ( Figure 3 , green stars) are not all conservative in RK9NH, which suggests an even broader substrate specificity. It is also evident that RK9NH differs from Lactobacillus buchneri LBK78 nucleoside hydrolase, although these two enzymes share the same function.…”

“…Most unexpectedly, RK9NH is not similar to a 2′- O -methylribonucleoside hydrolase from Lactobacillus buchneri (see Figure 3 ). This enzyme was discovered recently [ 27 ] and is already being used for the enzymatic synthesis of 2′- O -methylribonucleosides [ 28 ]. The phylogenetic analysis placed the RK9NH and the 2′- O -methylribonucleoside hydrolase from L. buchneri enzymes into separate groups ( Figure 2 ).…”

“… Multiple amino acid sequence alignment of nucleoside hydrolases. RihB: pyrimidine-specific ribonucleoside hydrolase from E. coli [ 39 ]; RihA: pyrimidine-specific ribonucleoside hydrolase from E. coli [ 33 ]; RihC: non-specific ribonucleoside hydrolase from E. coli [ 33 ]; LBK78: 2′- O -methylribonucleoside-specific nucleoside hydrolase from Lactobacillus buchneri LBK78 [ 27 ]; RK9NH: nucleoside hydrolase discovered in the metagenomic libraries. Highly similar residues are in red and framed in blue, strictly identical residues are in white on a red background.…”

“…The ribose 2′- O -methylation increases the hydrophobicity of nucleotides and protects them against the action of nucleases [ 25 , 26 ], which is a desired trait, especially for, e.g., DNA aptamers [ 24 ]. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding enzymes involved in the metabolism of 2′- O -alkyl nucleosides [ 27 , 28 , 29 ]. The presented work focuses on the cellular degradation of 2′- O -methyluridine that is found naturally in rRNA, snRNA, snoRNA and tRNA of Archaea , Eubacteria , and Eukaryota [ 1 ].…”

Ribonucleoside Hydrolases–Structure, Functions, Physiological Role and Practical Uses

“…Examples of reactions are shown in Figure 1 and Figure 2 . Hydrolases of this type are found in a wide variety of organisms: bacteria [ 21 , 34 , 45 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 ], plants [ 23 , 24 , 25 , 26 , 27 ], yeast [ 28 , 38 ], fungi [ 29 , 32 ], metazoa [ 34 , 35 , 37 ], and also some protozoa and archaea [ 53 ].…”