CYP51 from Trypanosoma brucei Is Obtusifoliol-Specific

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Abstract
New isoforms of CYP51 (sterol 14α-demethylase), an essential enzyme in sterol biosynthesis and primary target of azole antimycotic drugs, are found in pathogenic protists, Trypanosoma brucei (TB), T. vivax, T. cruzi, and Leishmania major. The sequences share ∼80% amino acid identity and are ∼25% identical to sterol 14α-demethylases from other biological kingdoms. Differences of residues conserved throughout the rest of the CYP51 family that align with the BC-loop and helices F and G of CYP51 from Mycobacterium tuberculosis (MT)) imply possible alterations in the topology of the active site cavity of the protozoan enzymes. CYP51 and cytochrome P450 reductase (CPR) from TB were cloned, expressed in Escherichia coli, and purified. The P450 has normal spectral features (including absolute absorbance, carbon monoxide, and ligand binding spectra), is efficiently reduced by TB and rat CPR but demonstrates altered specificity in comparison with human CYP51 toward three tested azole inhibitors, and contrary to the human, Candida albicans, and MT isoforms, reveals profound substrate preference toward obtusifoliol (turnover 5.6 min-1). It weakly interacts with the other known CYP51 substrates; slow lanosterol conversion predominantly produces the 14α-carboxyaldehyde intermediate. Although obtusifoliol specificity is typical for plant isoforms of CYP51, the set of sterol biosynthetic enzymes in the protozoan genomes together with available information about sterol composition of kinetoplastid cells suggest that the substrate preference of TBCYP51 may reflect a novel sterol biosynthetic pathway in Trypanosomatidae.

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