Trypanosoma brucei brucei: thymine 7-hydroxylase-like proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Parasitology

Background:

• Trypanosoma brucei brucei possesses genes predicted to encode Fe(II)- and alpha-ketoglutarate-dependent enzymes.
• These enzymes are homologous to fungal thymine 7-hydroxylase.
• Differential gene expression and protein levels exist between parasite life stages.

Purpose of the Study:

• To investigate the enzymatic activity of two T. brucei brucei genes homologous to fungal thymine 7-hydroxylase.
• To determine if these enzymes are involved in thymine salvage pathways in the parasite.

Main Methods:

• Gene cloning and protein purification from Escherichia coli.
• Biochemical assays to test enzyme activity with various substrates and cofactors (Fe(II), alpha-ketoglutarate).
• Western blot analysis to assess protein levels in different parasite life stages.

Main Results:

• Purified proteins confirmed to bind Fe(II) and alpha-ketoglutarate, indicating proper folding.
• No thymine 7-hydroxylase activity was detected in vitro with thymine, thymidine, or other potential substrates.
• No thymine 7-hydroxylase activity was found in extracts from procyclic or bloodstream forms of T. brucei brucei.

Conclusions:

• The studied T. brucei brucei proteins, despite binding necessary cofactors, do not exhibit thymine 7-hydroxylase activity.
• These enzymes are unlikely to play a role in the thymine salvage pathway in this parasite.
• The actual functions of these Fe(II)- and alpha-ketoglutarate-dependent enzymes in T. brucei brucei remain to be elucidated.