To determine the orthology of sterol 14-demethylase (P45014DM), the only known P450 enzyme distributed widely in eukaryotes with a conserved metabolic role, the full-length amino acid sequences of rat and human P45014DMs were determined from the cloned cDNA sequences, and compared with those of the corresponding fungal proteins (CYP51). The amino acid identity value between given pairs of P45014DMs ranged from 93% (human/rat) to 39% (human or rat/Saccharomyces cerevisiae). All the P45014DMs formed a single cluster in a phylogenetic tree constructed from representative P450 protein sequences currently available. The nearest neighbors to the P45014DM cluster in the phylogenetic tree were CYP7 (cholesterol 7 alpha-hydroxylase) and CYP8 (prostacyclin synthase), and the divergence point of fungal and mammalian P45014DMs was clearly more recent than that of P45014DM and CYP7/CYP8. These lines of evidence show that fungal and mammalian P45014DMs are really orthologous. This is the first example of orthologous P450s occurring in distinct kingdoms. P45014DM may be an ancient P450 which arose before the divergence of major eukaryotic branches and has been conserved throughout evolution. The amino acid identity value (93%) between human and rat P45014DMs was comparable to those observed for some housekeeping enzymes. In addition, a processed pseudogene of P45014DM was found in a rat genomic DNA library, suggesting the expression of P45014DM in germ line cells. These facts suggest that P45014DM may be a housekeeping enzyme essential for the viability of mammals.

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Phylogenetic analyses based on protein sequence data indicated that sterol 14-demethylase P450 (CYP51) and bacterial CYP51-like protein were joined into a distinctive evolutionary cluster, CYP51 cluster, within the CYP protein superfamily. The most probable branch topology of the CYP51 phylogenetic tree was (bacteria, (plants, (fungi, mammals))), which is comparable to the phylogeny of major kingdoms of living matter, suggesting that CYP51 has been conserved from the era of prokaryotic evolution. This may be strong evidence supporting the prokaryotic origin of P450. Structure of flanking regions and the number and insertion sites of introns are quite different between mammalian and fungal CYP51s. This fact indicates that different mechanisms are operative in evolution of protein sequences and gene structures. CYP51 is the first example violating the well-documented rule that the basic structure of a gene, including intron insertion sites, is well conserved in each P450 family. One CYP51 processed a pseudogene was found in rat genome. Nonsynonymous nucleotide divergence observed between the pseudogene and CYP51 cDNA was less than one-fifth of the synonymous divergence. This unusually low rate of nonsynonymous nucleotide changes in the pseudogene suggests that it may be derived from another CYP51, which might have been active for a significant duration in the past.

The amino acid residues affecting the function of rat sterol 14-demethylase P450 (CYP51) were examined by means of point mutation. Forty-five mutants with respect to 27 amino acid sites were constructed and expressed in Escherichia coli. Substitution of highly conserved Y131, E369, R372, or R382 decreased the expression of CYP51 protein, indicating some structural importance of these residues. Substitution of H314, T315, or S316 caused considerable effects on the catalytic activity, and T315 was identified as the "conserved threonine" of CYP51. H314 was important for maintenance of the activity of CYP51 and was a characteristic residue of this P450, because the position corresponding to this residue is occupied by an acidic amino acid in most other P450 species. A144 was identified as a residue affecting the interaction of CYP51 with ketoconazole. Substitution of A144 with I, which occupies the corresponding position in fungal CYP51, enhanced the ketoconazole susceptibility of rat CYP51 with little change in the catalytic activity, indicating an important role of this residue in determination of the ketoconazole susceptibility of CYP51. Alteration of the catalytic activity was caused by the substitution at some other sites, whereas substitution of a few highly conserved amino acids caused little alteration of the activity of CYP51.

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