Characterization and catalytic properties of the sterol 14α-demethylase from Mycobacterium tuberculosis

Abstract

Sterol 14␣-demethylase encoded by CYP51 is a mixed-function oxidase involved in sterol synthesis in eukaryotic organisms. Completion of the Mycobacterium tuberculosis genome project revealed that a protein having homology to mammalian 14␣-demethylases might be present in this bacterium. Using genomic DNA from mycobacterial strain H 37 Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14␣-demethylase. Expression of the M. tuberculosis CYP51 gene in Escherichia coli yields a P450, which, when purified to homogeneity, has the predicted molecular mass, ca. 50 kDa on SDS͞PAGE, and binds both sterol substrates and azole inhibitors of P450 14␣-demethylases. It catalyzes 14␣-demethylation of lanosterol, 24,25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC͞MS and 1 H NMR analysis. Both f lavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14␣-methyl group and ⌬ 8(9) -bond were established by comparing binding of pairs of sterol substrate that differed in a single molecular feature, e.g., cycloartenol paired with lanosterol. These substrate requirements are similar to those established for plant and animal P450 14␣-demethylases. From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.

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

“…In the reduced CO-complexes bacterial enzymes show maximum at 450 nm, while in the spectra of the eukaryotic orthologs the maximum is shifted to 446-447 nm, which might be connected with the formation of aggregates. Similar to other microsomal P450s, the eukaryotic forms of CYP51 require cytochrome P450 reductase as an electron donor partner; bacterial CYP51 from M. tuberculosis, M. smegmatis and M. capsulatus can be enzymatically reduced by flavodoxin/ flavodoxin reductase and ferredoxin/ferredoxin reductase systems [14,37].…”

“…Amongst them 9 are microsomal, eukaryotic enzymes (including 3 mammalian: rat [7], pig [32], and human [33]; 1 plant: S. bicolor [12], 3 fungal: 2 from yeasts (S. cerevisiae [8], Candida albicans [34]), and one from filamentous fungus Ustilago maydis [35] and 2 trypanosomal (Trypanosoma brucei [27] and T. cruzi [36]) while the other 3 are water soluble bacterial orthologs (from M. tuberculosis [14], M. smegmatis [37], and a fusion CYP51-ferredoxin protein from Methylococcus capsulatus [38]). …”

“…In the reduced CO-complexes bacterial enzymes show maximum at 450 nm, while in the spectra of the eukaryotic orthologs the maximum is shifted to 446-447 nm, which might be connected with the formation of aggregates. Similar to other microsomal P450s, the eukaryotic forms of CYP51 require cytochrome P450 reductase as an electron donor partner; bacterial CYP51 from M. tuberculosis, M. smegmatis and M. capsulatus can be enzymatically reduced by flavodoxin/ flavodoxin reductase and ferredoxin/ferredoxin reductase systems [14,37].Contrary to drug metabolizing P450s, which can accommodate a vast variety of structures, CYP51 has very narrow substrate specificity. The set of substrates is limited to the five naturally occurring 14α-methylsterols (lanosterol, 24, 25-dihydrolanosterol, 24-methylenedihydrolanosterol, obtusifoliol and 4β-desmethyllanosterol (norlanosterol [36]) (see Figure 2)).…”

“…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.…”

Quantitation of lanosterol and its major metabolite FF-MAS in an inhibition assay of CYP51 by azoles with atmospheric pressure photoionization based LC-MS/MS

“…Lanosterol and its metabolites were predominantly quantitated directly or after derivatization by gas chromatography mass spectrometry (GC-MS) [7,8]. If amounts of about 1 ng of each steroid were available, lanosterol and FF-MAS could be separated by reversed phase high performance liquid chromatography (HPLC) and detected using a diode array detector [9].…”