Functional analyses of dipeptide and pentapeptide insertions on Theileria annulata enolase by site-directed mutagenesis and in silico approaches

Biochim Biophys Acta Proteins Proteom. Jul-Aug 2019;1867(7-8):732-739. doi: 10.1016/j.bbapap.2019.05.006. Epub 2019 May 16.


Theileria annulata enolase (TaENO) could be assessed as a druggable target for tropical theileriosis treatment. The parasite enzyme plays an important role in many cellular functions and carries some structural differences like dipeptide (262EK263) and pentapeptide (103EWGYC107) insertions from the host enzyme, Bos taurus enolase. In this study, the functional effects of these insertions on TaENO activity were analyzed by in vitro site-directed mutagenesis and in silico molecular docking analyses for the first time in the literature. In vitro results showed that, although the deletion of the pentapeptide insertion (TaENOΔEWGYC) reduced the enzyme activity slightly, the removal of the dipeptide insertion (TaENOΔEK) halted it. Also, molecular docking results revealed that the deletion of these insertions affected the substrate binding affinity of the mutant enzymes. The active site of TaENOΔEK exhibited a small decrease of substrate binding affinity compared to the active site of TaENOΔEWGYC relative to the wild type TaENO. Although we conclude that both regions could be evaluated as possible drug-binding sites to inhibit TaENO in further studies, these results indicate that the dipeptide insertion could be a more promising drug binding site than the pentapeptide insertion.

Keywords: Enolase; Enzyme activity; Molecular docking; Site-directed mutagenesis; Theileria annulata.

MeSH terms

  • Animals
  • Catalytic Domain / genetics
  • Cattle
  • Dipeptides / chemistry*
  • Dipeptides / genetics
  • Molecular Docking Simulation*
  • Mutagenesis, Site-Directed*
  • Phosphopyruvate Hydratase / chemistry*
  • Phosphopyruvate Hydratase / genetics
  • Protozoan Proteins / chemistry*
  • Protozoan Proteins / genetics
  • Substrate Specificity / genetics
  • Theileria annulata / enzymology*
  • Theileria annulata / genetics


  • Dipeptides
  • Protozoan Proteins
  • Phosphopyruvate Hydratase