The structural and functional properties of the cytochrome P450 superfamily have been subject to extensive diversification over the course of evolution. Recent estimates indicate that there are currently 10 classes and 267 families of CYP proteins. 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.
Although CYP51's mode of action has been well conserved, the protein's sequence varies considerably between biological kingdoms. CYP51 sequence comparisons between kingdoms reveal only a 22-30% similarity in amino acid composition.
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. 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.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. Finally, the active site contains a hemeprosthetic group in which the iron is tethered to the sulfur atom on a conserved cysteine residue. This group also binds diatomic oxygen at the sixth coordination site, which is eventually incorporated onto the substrate.
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). 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. The aldehyde then departs as formic acid and a double bond is simultaneously introduced to yield the demethylated product.
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