Sterol 14-Demethylase P450 (P45014DM*) Is One of the Most Ancient and Conserved P450 Species

Abstract

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.

Rapid P450 Heme Iron Reduction by Laser Photoexcitation of Mycobacterium tuberculosis CYP121 and CYP51B1

“…Rates of decay from single wavelength data were consistent with the values determined from PDA analysis, at 0.72 Ϯ 0.02 s Ϫ1 . As has been observed previously, the Fe II -CO complex of CYP51B1 collapses from the P450 form to the P420 form over a period of several minutes (3,16). At pH 7.0 and 25°C, the rate was re-determined by following the spectral collapse and fitting data to a single exponential function.…”

“…EPR data also demonstrated a thiolate-coordinated P450 heme iron in the substrate-free enzyme (4,16). However, the Fe II -CO complex collapses from the P450 to a P420 form over a period of minutes, and the Fe II form also has a spectrum typical of a thiol-coordinated heme iron and comparable with thiolcoordinated heme iron in a H93G mutant of sperm whale myo- globin (3,4,16). Thus, there are considerable differences between the properties of the two Mtb P450s CYP121 and CYP151B1.…”

“…Mycobacterium tuberculosis (Mtb), 3 the bacterium that causes tuberculosis in humans, has made a dramatic global resurgence in recent years. The World Health Organization estimates that one-third of the human population is infected with the Mtb bacillus (1,2).…”

“…One of the most surprising features was the presence of 20 different genes encoding cytochrome P450 (CYP) enzymes. The repertoire of CYP enzymes included the first prokaryotic example of a sterol demethylase P450 (CYP51) (3,4). Eukaryotic CYP51 enzymes catalyze the oxidative demethylation of sterol substrates leading to removal of the 14␣-methyl group.…”

A different function for a member of an ancient and highly conserved cytochrome P450 family: From essential sterols to plant defense

“…The CYP51 sterol demethylases are regarded as the most ancient cytochrome P450 family. They are highly conserved across the animal, fungal, and plant kingdoms and are known only to have a single strictly conserved function, in the synthesis of essential sterols (10)(11)(12)(13). AsCYP51H10 belongs to a unique subfamily of divergent plant CYP51 enzymes (CYP51H) that until now has been defined only by rice sequences of unknown function (11).…”