The structural and functional properties of the cytochrome P450 superfamily have been subject to extensive diversification over the course of evolution.[7] Recent estimates indicate that there are currently 10 classes and 267 families of CYP proteins.[8] It is believed that 14α-demethylase or CYP51 diverged early in the cytochrome's evolutionary history and has preserved its function ever since; namely, the removal of the 14α-methyl group from sterol substrates.[7]
Although CYP51's mode of action has been well conserved, the protein's sequence varies considerably between biological kingdoms.[9] CYP51 sequence comparisons between kingdoms reveal only a 22-30% similarity in amino acid composition.[10]
Structure
Structure of lanosterol 14α-demethylase (CYP51), as identified by Podust et al.
Although the structure of 14α-demethylase may vary substantially from one organism to the next, sequence alignment analysis reveals that there are six regions in the protein that are highly conserved in eukaryotes.[10] These include residues in the B' helix, B'/C loop, C helix, I helix, K/β1-4 loop, and β-strand 1-4 that are responsible for forming the surface of the substrate binding cavity.[7]Homology modeling reveals that substrates migrate from the surface of the protein to the enzyme's buried active site through a channel that is formed in part by the A' alpha helix and the β4 loop.[11][12] Finally, the active site contains a hemeprosthetic group in which the iron is tethered to the sulfur atom on a conserved cysteine residue.[10] This group also binds diatomic oxygen at the sixth coordination site, which is eventually incorporated onto the substrate.[10]
Mechanism
Three-step demethylation of lanosterol, mediated by lanosterol 14α-demethylase.
The enzyme-catalyzed demethylation of lanosterol is believed to occur in three steps, each of which requires one molecule of diatomic oxygen and one molecule of NADPH (or some other reducing equivalent).[13] During the first two steps, the 14α-methyl group undergoes typical cytochrome monooxygenation in which one oxygen atom is incorporated by the substrate and the other is reduced to water, resulting in the sterol's conversion to a carboxyalcohol and then a carboxyaldehyde.[10] The aldehyde then departs as formic acid and a double bond is simultaneously introduced to yield the demethylated product.[10]
^ abcBecher R, Wirsel SG (August 2012). "Fungal cytochrome P450 sterol 14α-demethylase (CYP51) and azole resistance in plant and human pathogens". Applied Microbiology and Biotechnology. 95 (4): 825–40. doi:10.1007/s00253-012-4195-9. PMID22684327. S2CID17688962.
^Hannemann F, Bichet A, Ewen KM, Bernhardt R (March 2007). "Cytochrome P450 systems--biological variations of electron transport chains". Biochimica et Biophysica Acta (BBA) - General Subjects. 1770 (3): 330–44. doi:10.1016/j.bbagen.2006.07.017. PMID16978787.
Aoyama Y, Yoshida Y (August 1991). "Different substrate specificities of lanosterol 14a-demethylase (P-45014DM) of Saccharomyces cerevisiae and rat liver for 24-methylene-24,25-dihydrolanosterol and 24,25-dihydrolanosterol". Biochemical and Biophysical Research Communications. 178 (3): 1064–71. doi:10.1016/0006-291X(91)91000-3. PMID1872829.
Aoyama Y, Yoshida Y (March 1992). "The 4 beta-methyl group of substrate does not affect the activity of lanosterol 14 alpha-demethylase (P-450(14)DM) of yeast: difference between the substrate recognition by yeast and plant sterol 14 alpha-demethylases". Biochemical and Biophysical Research Communications. 183 (3): 1266–72. doi:10.1016/S0006-291X(05)80327-4. PMID1567403.
Alexander K, Akhtar M, Boar RB, McGhie JF, Barton DH (1972). "The removal of the 32-carbon atom as formic acid in cholesterol biosynthesis". Journal of the Chemical Society, Chemical Communications (7): 383. doi:10.1039/C39720000383.
