CYP51 from Trypanosoma cruzi

Abstract

A potential drug target for treatment of Chagas disease, sterol 14␣-demethylase from Trypanosoma cruzi (TCCYP51), was found to be catalytically closely related to animal/fungi-like CYP51. Contrary to the ortholog from Trypanosoma brucei (TB), which like plant CYP51 requires C4-monomethylated sterol substrates, TCCYP51 prefers C4-dimethylsterols. Sixty-six CYP51 sequences are known from bacteria to human, their sequence homology ranging from ϳ25% between phyla to ϳ80% within a phylum. TC versus TB is the first example of two organisms from the same phylum, in which CYP51s (83% amino acid identity) have such profound differences in substrate specificity. Substitution of animal/fungi-like Ile 105 in the B helix to Phe, the residue found in this position in all plant and the other six CYP51 sequences from Trypanosomatidae, dramatically alters substrate preferences of TCCYP51, converting it into a more plant-like enzyme. The rates of 14␣-demethylation of obtusifoliol and its 24-demethyl analog 4␣-,4␣-dimethylcholesta-8,24-dien-3␤-ol(norlanosterol) increase 60-and 150-fold, respectively. Turnover of the three 4,4-dimethylated sterol substrates is reduced ϳ3.5-fold. These catalytic properties correlate with the sterol binding parameters, suggesting that Phe in this position provides necessary interactions with C4-monomethylated substrates, which Ile cannot. The CYP51 substrate preferences imply differences in the post-squalene portion of sterol biosynthesis in TC and TB. The phyla-specific residue can be used to predict preferred substrates of new CYP51 sequences and subsequently for the development of new artificial substrate analogs, which might serve as highly specific inhibitors able to kill human parasites.

Structural Insights into Inhibition of Sterol 14α-Demethylase in the Human Pathogen Trypanosoma cruzi

“…TC14DM Gene Cloning and Modifications-The gene-encoding 14DM was amplified from T. cruzi genomic DNA with the addition of a His 6 tag at the protein C terminus and cloned into a pCW expression plasmid as described previously (29). For crystallization purposes, the N-terminal transmembrane domain upstream of Pro 32 was replaced with MAKKTSSKGKL- (28) in the construct used for co-crystallization with fluconazole and VNF (construct 1) and with MAKKT-(5Ј-ATGGTCAAGAAAACG-3Ј) in the complex with posaconazole (construct 2).…”

“…Purification and Crystallization of TC14DM-Expression and purification of the full-length TC and human 14DMs was reported elsewhere (29,30). The N-terminal truncation resulted in the increased expression levels of TC14DM, up to 0.7 and 1.5 mol/liter for constructs 1 and 2, respectively.…”

“…The N-terminal truncation resulted in the increased expression levels of TC14DM, up to 0.7 and 1.5 mol/liter for constructs 1 and 2, respectively. Truncated protein was purified following the same steps as the full-length TC14DM (29), including nickel-nitrilotriacetic acid (Qiagen) and ion-exchange chromatography (Q-Sepharose followed by SP-Sepharose, Amersham Biosciences), except that Triton X-100 was replaced by 0.048 mM n-tridecyl-␤-D-maltoside (Anatrace). The initial screening of crystallization conditions was performed using Hampton Research crystallization kits.…”

Conformational dynamics in the F/G segment of CYP51 from Mycobacterium tuberculosis monitored by FRET

“…Because of this correlation between the amplitude of the spectral response and CYP51 enzymatic activity [23] we calculated the percentage of substrate-bound MTCYP51 from the high-spin form content. Contrary to many P450s which exist in the equilibrium of the high-and low-spin forms regardless of the presence of substrate, MTCYP51 [23,40] (as well as the eukaryotic CYP51 orthologs the authors are familiar with [13,38,39]) and never show any alterations in the spin-state as a result of changes in the environment (such as temperature, or buffer composition, pH, presence of glycerol or a detergent, etc. ); water molecule coordinated to the heme iron (which keeps MTCYP51 in the low-spin state instead of the high-/low-spin state equilibrium in the absence of the substrate) can be seen in the structure of ligand-free MTCYP51 [1h5z] [41].…”

“…It is required for sterol biosynthesis and catalyzes demethylation of 5 natural sterol substrates which have very subtle structural differences [13]. In eukaryotes this P450 is localized in the endoplasmic reticulum and in prokaryotes it is a soluble protein.…”