Structural and biochemical characterization of the nucleoside hydrolase from C. elegans reveals the role of two active site cysteine residues in catalysis

Protein Sci. 2017 May;26(5):985-996. doi: 10.1002/pro.3141. Epub 2017 Mar 6.


Nucleoside hydrolases (NHs) catalyze the hydrolysis of the N-glycoside bond in ribonucleosides and are found in all three domains of life. Although in parasitic protozoa a role in purine salvage has been well established, their precise function in bacteria and higher eukaryotes is still largely unknown. NHs have been classified into three homology groups based on the conservation of active site residues. While many structures are available of representatives of group I and II, structural information for group III NHs is lacking. Here, we report the first crystal structure of a purine-specific nucleoside hydrolase belonging to homology group III from the nematode Caenorhabditis elegans (CeNH) to 1.65Å resolution. In contrast to dimeric purine-specific NHs from group II, CeNH is a homotetramer. A cysteine residue that characterizes group III NHs (Cys253) structurally aligns with the catalytic histidine and tryptophan residues of group I and group II enzymes, respectively. Moreover, a second cysteine (Cys42) points into the active site of CeNH. Substrate docking shows that both cysteine residues are appropriately positioned to interact with the purine ring. Site-directed mutagenesis and kinetic analysis proposes a catalytic role for both cysteines residues, with Cys253 playing the most prominent role in leaving group activation.

Keywords: N-ribohydrolase; SAXS; X-ray crystallography; enzyme mechanism; leaving group activation; nucleoside hydrolase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caenorhabditis elegans / enzymology*
  • Caenorhabditis elegans / genetics
  • Caenorhabditis elegans Proteins / chemistry*
  • Caenorhabditis elegans Proteins / genetics
  • Catalytic Domain
  • Crystallography, X-Ray
  • Cysteine
  • N-Glycosyl Hydrolases / chemistry*
  • N-Glycosyl Hydrolases / genetics


  • Caenorhabditis elegans Proteins
  • N-Glycosyl Hydrolases
  • Cysteine

Associated data

  • PDB/3FZ0
  • PDB/3t8i
  • PDB/2mas
  • PDB/3fz0
  • PDB/4i73
  • PDB/1kic
  • PDB/3B9X
  • PDB/2FF2
  • PDB/2MAS
  • PDB/5MJ7