Biodiversity of CYP51 in trypanosomes

Area of Science:

• Biochemistry
• Molecular Biology
• Parasitology

Background:

• Sterol 14alpha-demethylases (CYP51) are crucial enzymes in sterol biosynthesis across all kingdoms of life.
• Plant CYP51s metabolize C-4-monomethylated obtusifoliol, while animal/fungal CYP51s process C-4-dimethylated lanosterol derivatives.
• CYP51 from Trypanosoma brucei (TB) is an O-specific enzyme, unique for non-photosynthetic organisms.

Purpose of the Study:

• To investigate the substrate specificity of CYP51 from Trypanosoma cruzi (TC) and its comparison to TB CYP51.
• To elucidate the structural basis for the differing substrate preferences between TC and TB CYP51.
• To explore the evolutionary implications of these differences in sterol metabolism.

Main Methods:

• Comparative analysis of amino acid sequences between TB CYP51 and TC CYP51.
• Site-directed mutagenesis of TC CYP51 to replace Ile105 with Phenylalanine.
• Enzyme kinetics assays using various sterol substrates, including obtusifoliol (O) and norlanosterol (N).

Main Results:

• TC CYP51 exhibits a strong preference for C-4-dimethylated sterols, contrasting with the O-specificity of TB CYP51, despite high sequence identity.
• Mutating Ile105 to Phenylalanine in TC CYP51 (creating a plant/TB-like residue) dramatically enhanced its ability to metabolize obtusifoliol (O).
• The mutant TC CYP51 showed over 100-fold increased binding and turnover for norlanosterol (N), a known TB CYP51 substrate found in vivo.

Conclusions:

• Norlanosterol (N) represents a non-conventional substrate for CYP51, preferentially utilized by TB and potentially other Trypanosomatidae.
• The functional similarity of TC CYP51 to animal/fungal orthologues is attributed to evolutionary convergence, including the F105I mutation.
• Distinct sterol production pathways have evolved in T. cruzi and T. brucei, driven by differences in their CYP51 enzymes.