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J. Biol. Chem., Vol. 281, Issue 6, 3577-3585, February 10, 2006

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CYP51 from Trypanosoma cruzi

A PHYLA-SPECIFIC RESIDUE IN THE B' HELIX DEFINES SUBSTRATE PREFERENCES OF STEROL 14-DEMETHYLASE^*

Galina I. Lepesheva¹, Natalia G. Zaitseva, W. David Nes, Wenxu Zhou, Miharu Arase, Jialin Liu, George C. Hill^¶, and Michael R. Waterman

From the Departments of Biochemistry and ^¶Microbiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409

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 25% between phyla to 80% 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 Ile¹⁰⁵ 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 3.5-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.

Received for publication, September 20, 2005 , and in revised form, November 29, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY856083 [GenBank] .

^* This work was supported by National Institutes of Health Grants GM067871 and ES00267-32 (to M. R. W.) and GM63477 (to W. D. N.), American Heart Association Grant 0535121N (to G. I. L.), and Welch Foundation Grant D-1276 to W. D. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. Tel.: 615-343-1373; Fax: 615-322-4349; E-mail: galina.i.lepesheva{at}vanderbilt.edu.

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