8UCH

Thermophilic RNA Ligase from Palaeococcus pacificus K92A + ATP

8UCH の概要

• エントリーDOI: 10.2210/pdb8uch/pdb
• 分子名称: ATP dependent DNA ligase, ADENOSINE-5'-TRIPHOSPHATE, GLYCEROL, ... (7 entities in total)
• 機能のキーワード: rna ligase, thermophilic, archaea, rnl3, nucleotidyl-transferase, ligase
• 由来する生物種: Palaeococcus pacificus DY20341
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 48078.71

構造登録者

Rousseau, M.D.,Hicks, J.L.,Oulavallickal, T.,Williamson, A.,Arcus, V.L.,Patrick, M.W. (登録日: 2023-09-26, 公開日: 2024-02-21, 最終更新日: 2024-05-08)

主引用文献

Rousseau, M.,Oulavallickal, T.,Williamson, A.,Arcus, V.,Patrick, W.M.,Hicks, J.
Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus.
Nucleic Acids Res., 52:3924-3937, 2024

PubMed Abstract: RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl). PpaRnl exhibited significant substrate adenylation activity but low ligation activity across a range of oligonucleotide substrates. Mutation of Lys92 in motif I to alanine, resulted in an enzyme that lacked adenylation activity, but demonstrated improved ligation activity with pre-adenylated substrates (ATP-independent ligation). Subsequent structural characterisation revealed that in this mutant enzyme Lys238 was found in two alternate positions for coordination of the phosphate tail of ATP. In contrast mutation of Lys238 in motif V to glycine via structure-guided engineering enhanced ATP-dependent ligation activity via an arginine residue compensating for the absence of Lys238. Ligation activity for both mutations was higher than the wild-type, with activity observed across a range of oligonucleotide substrates with varying sequence and secondary structure.

PubMed: 38421610
DOI: 10.1093/nar/gkae149

実験手法

X-RAY DIFFRACTION (2.14 Å)