Mutagenesis of the novel Hericium erinaceus ribonuclease, RNase He1, reveals critical responsible residues for enzyme stability and activity

Biol Pharm Bull. 2014;37(11):1843-7. doi: 10.1248/bpb.b14-00553.

Abstract

Here, we determined the sequence of a cDNA encoding a guanylic acid-specific ribonuclease (RNase He1) from Hericium erinaceus that exhibits high sequence identity (59%) with RNase Po1, an enzyme with anti-cancer activity and which is found in Pleurotus ostreatus. RNase He1 and RNase Po1 have similar structures and heat stabilities; hence, RNase He1 may also have potential as an anti-cancer agent. Therefore, we initiated structure-function studies to further characterize the enzyme. Based on the RNase Po1 structure, RNase He1 is predicted to form 3 disulfide bonds involving Cys7-Cys98, Cys5-Cys83, and Cys47-Cys81 linkages. The Cys5Ala mutant exhibited no RNase activity, whereas the Cys81Ala mutant retained RNase activity, but had reduced heat stability. Therefore, the Cys5-Cys83 bond in RNase He1 is essential for the structure of the RNase active site region. Similarly, the Cys47-Cys81 bond helps maintain the conformational stability of the active site region, and may contribute to the greater heat stability of RNase He1.

MeSH terms

  • Basidiomycota / enzymology*
  • Cell Proliferation / drug effects
  • Cysteine / genetics
  • Enzyme Stability
  • HL-60 Cells
  • Humans
  • Mutagenesis
  • RNA / metabolism
  • Ribonucleases* / chemistry
  • Ribonucleases* / genetics
  • Ribonucleases* / metabolism
  • Ribonucleases* / pharmacology

Substances

  • RNA
  • Ribonucleases
  • Cysteine