Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology

Bellamine, Aouatef; Lepesheva, Galina I.; Waterman, Michael R.

doi:10.1194/jlr.m400239-jlr200

Cited by 53 publications

(48 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, at saturating concentrations of eburicol, Ͻ10% of the MgCYP51 molecules changed spin state from low to high spin. This relatively low degree of spin state conversion induced by substrate binding has also been observed with other CYP51 enzymes (3,7,16,25), with spin state changes usually not exceeding 10%.…”

Section: Resultsmentioning

confidence: 81%

Mechanism of Binding of Prothioconazole to Mycosphaerella graminicola CYP51 Differs from That of Other Azole Antifungals

Parker

Warrilow

Cools

et al. 2011

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

Section: Resultsmentioning

confidence: 81%

Mechanism of Binding of Prothioconazole to Mycosphaerella graminicola CYP51 Differs from That of Other Azole Antifungals

Parker

Warrilow

Cools

et al. 2011

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…tuberculosis CYP51 is present in this position in all bacterial and fungal CYP51 but in the other species this position is occupied by Y. Mutation of F89 inactivates M. tuberculosis CYP51 but substitution of the corresponding Y in human or C. albicans orthologs has no effect [47] on their activities and interaction with the substrate. Similarly, mutation D90A inactivates M. tuberculosis CYP51 but the D132A mutant of the human ortholog partially retains its sterol 14α-demethylase activity.…”

Section: Cyp51 Signature Structural Basis Of Conservation In the Cypmentioning

confidence: 99%

“…The effect of the substitution of two other residues F89 [47] and D90 [42] in this loop is probably phylum-specific. F corresponding to F89 in M.…”

Section: Cyp51 Signature Structural Basis Of Conservation In the Cypmentioning

confidence: 99%

Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

Lepesheva

Waterman

2007

Biochimica et Biophysica Acta (BBA) - General Subjects

Self Cite

408

331

View full text Add to dashboard Cite

SummaryThe CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 evolution are discussed and the major approaches for CYP51 inhibition are summarized.Keywords sterol 14α-demethylase (CYP51); sterol biosynthesis; substrate preferences; catalytic activity; inhibition Family overviewSterol 14α-demethylation as a general part of sterol biosynthetic pathways in eukaryotes [1] has been known and studied for more than 30 years [2][3][4][5][6][7]. The enzyme catalyzing this reaction was first purified from yeast in 1984 (Sacharomyces cerevisiea [8]), and following determination of its primary structure [9] the cytochrome P450 sterol 14α-demethylases were placed into the CYP51 family, a number reserved for fungal sequences [10]. In 1986 the orthologous mammalian P450 was purified from rat liver microsomes [11], in 1996 the first sterol 14α-demethylase was found in plants (Sorghum bicolor [12]), and in 2000 the orthologous nature of a CYP51-like gene [13] from Mycobacterium tuberculosis to eukaryotic CYP51s was confirmed [14].Currently the CYP51 family joins proteins found in 82 organisms from all biological kingdoms. In addition, several plants and fungi contain multiple CYP51 genes (e.g. rice (10), black oats (2), tobacco (2), Arabidopsis thaliana (2), Fuzarium graminearum (3) or Aspergillus species: A. fumigatus (2), A.nidulans (2), A. orizae (3)). As a result, the number of known CYP51 sequences exceeds 100. The reasons for the existence of homologous CYP51 genes in the same species or their precise functions remain unknown, though it was reported that only one of the two CYP51 genes from A. thaliana (CYP51A2) is functional, while the other is an expressed pseudogene [15]. Mammalian genomes contain only one CYP51 gene yet sometimes † Corresponding author: Michael R. Waterman, Tel.: 615-343-1373; Fax: 615-322-4349; E-mail: michael.waterman@vanderbilt.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The average amino acid sequence identity in the CYP51 family is about 30%, varying from relatively high between the proteins from evolutionary closely related species (e.g. 95% in mammals) to lower values within the highly diverse kingdoms of lower...

show abstract

“…DHBP-BC-loop Interactions-A remarkable finding provided by the crystal structure of the CYP51 Mt -DHBP complex is elucidation of the function of residue Phe-89, which is important for substrate binding and conversion in CYP51 Mt (46). Well conserved Phe-89 (supplemental Fig.…”

Section: Binding Of Dhbp In the Activementioning

confidence: 99%

X-ray Structure of 4,4′-Dihydroxybenzophenone Mimicking Sterol Substrate in the Active Site of Sterol 14α-Demethylase (CYP51)

Eddine

Kries²,

Podust

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

A universal step in the biosynthesis of membrane sterols and steroid hormones is the oxidative removal of the 14␣-methyl group from sterol precursors by sterol 14␣-demethylase (CYP51). This enzyme is a primary target in treatment of fungal infections in organisms ranging from humans to plants, and development of more potent and selective CYP51 inhibitors is an important biological objective. Our continuing interest in structural aspects of substrate and inhibitor recognition in CYP51 led us to determine (to a resolution of 1.95 Å ) the structure of CYP51 from Mycobacterium tuberculosis (CYP51 Mt ) cocrystallized with 4,4-dihydroxybenzophenone (DHBP), a small organic molecule previously identified among top type I binding hits in a library screened against CYP51 Mt . The newly determined CYP51 Mt -DHBP structure is the most complete to date and is an improved template for three-dimensional modeling of CYP51 enzymes from fungal and prokaryotic pathogens. The structure demonstrates the induction of conformational fit of the flexible protein regions and the interactions of conserved Phe-89 essential for both fungal drug resistance and catalytic function, which were obscure in the previously characterized CYP51 Mt -estriol complex. DHBP represents a benzophenone scaffold binding in the CYP51 active site via a type I mechanism, suggesting (i) a possible new class of CYP51 inhibitors targeting flexible regions, (ii) an alternative catalytic function for bacterial CYP51 enzymes, and (iii) a potential for hydroxybenzophenones, widely distributed in the environment, to interfere with sterol biosynthesis. Finally, we show the inhibition of M. tuberculosis growth by DHBP in a mouse macrophage model.2 is a cytochrome P450 (P450, CYP) heme thiolate containing enzyme involved in biosynthesis of membrane sterols, including cholesterol in animals, ergosterol in fungi, and a variety of C24-modified sterols in plants and protozoa in most organisms in biological kingdoms from bacteria to animals (1). CYP51 has been a therapeutic target for several generations of azole antifungal agents including fluconazole, voriconazole, itraconazole, ravuconazole, and posaconazole (2). These drugs inhibit microbial growth by disrupting biosynthesis of ergosterol, a major component of fungal membrane. Protozoa share with fungi the requirement of ergosterol and ergosterol-related sterols for cell viability and proliferation (3). Inhibition of sterol biosynthesis has been proven to be effective in trypanosomatids (3-5) and Leishmania spp (6), which cause such tropical diseases as African sleeping sickness, Chagas disease, and leishmaniasis.Although mammalian CYP51 enzymes perform the same catalytic reaction (7) as their fungal and protozoan orthologs (1), they share relatively modest overall sequence identity (within 30%) with them. This accounts for the reduced sensitivity of mammalian CYP51 to azole and triazole drugs. Despite the lack of the full sterol biosynthetic pathway in Mycobacterium tuberculosis (8), and hence, de novo sterol biosynthesis...

show abstract

Fluconazole binding and sterol demethylation in three CYP51 isoforms indicate differences in active site topology

Cited by 53 publications

References 29 publications

Mechanism of Binding of Prothioconazole to Mycosphaerella graminicola CYP51 Differs from That of Other Azole Antifungals

Mechanism of Binding of Prothioconazole to Mycosphaerella graminicola CYP51 Differs from That of Other Azole Antifungals

Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

X-ray Structure of 4,4′-Dihydroxybenzophenone Mimicking Sterol Substrate in the Active Site of Sterol 14α-Demethylase (CYP51)

Contact Info

Product

Resources

About