Details

Autor(en) / Beteiligte

• Piotrowski, Yvonne
• Berg, Kristel
• Klebl, David Paul
• Leiros, Ingar
• Larsen, Atle Noralf

Titel

Characterization of an intertidal zone metagenome oligoribonuclease and the role of the intermolecular disulfide bond for homodimer formation and nuclease activity

Ist Teil von

• FEBS open bio, 2019-10, Vol.9 (10), p.1674-1688

Ort / Verlag

England: John Wiley & Sons, Inc

Erscheinungsjahr

2019

Link zum Volltext

Quelle

Wiley Online Library - AutoHoldings Journals

Beschreibungen/Notizen

• The gene encoding MG Orn has been identified from a metagenomic library created from the intertidal zone in Svalbard and encodes a protein of 184 amino acid residues. The mg orn gene has been cloned, recombinantly expressed in Escherichia coli, and purified to homogeneity. Biochemical characterization of the enzyme showed that it efficiently degrades short RNA oligonucleotide substrates of 2mer to 10mer of length and has an absolute requirement for divalent cations for optimal activity. The enzyme is more heat‐labile than its counterpart from E. coli and exists as a homodimer in solution. The crystal structure of the enzyme has been determined to a resolution of 3.15 Å, indicating an important role of a disulfide bridge for the homodimer formation and as such for the function of MG Orn. Substitution of the Cys110 residue with either Gly or Ala hampered the dimer formation and severely affected the enzyme's ability to act on RNA. A conserved loop containing His128‐Tyr129‐Arg130 in the neighboring monomer is probably involved in efficient binding and processing of longer RNA substrates than diribonucleotides. Here, we characterized an arctic metagenome oligoribonuclease and showed the importance of protein dimer formation for substrate binding and subsequent catalytic action. Our results suggest an important role of an intermolecular disulfide bond for the homodimer formation. We also show the in vitro ability of the dimeric oligoribonuclease to act on small as well as on ‘longer’ RNA oligos (5–10mer).