title: Pneumocystis carinii sterol 14α-demethylase activity in Saccharomyces cerevisiae erg11 knockout mutant: sterol biochemistry.
abstract: Pneumocystis carinii is an unusual fungus that can cause pneumonitis in immunosuppressed laboratory rats. Reactions in sterol biosynthesis are attractive targets for development of antimycotic drugs. A key enzyme in sterol biosynthesis is sterol 14α-demethylase (14DM), which is coded by the erg11 gene. Here we describe detailed sterol analysis of wild-type Saccharomyces cerevisiae and in an erg11 knockout mutant expressing either P. carinii or S. cerevisiae 14DM from a plasmid-borne cDNA. Sterols of the three strains were qualitatively and quantitatively analyzed using thin-layer chromatography, high-performance liquid chromatography, and gas-liquid chromatography and mass spectrometry and nuclear magnetic resonance spectroscopy. Biochemical evidence for functional complementation was provided by detecting the same major sterols in all three strains with ergosterol being by far the most abundant. A total of 25 sterols was identified, 16 of which were identified in all three strains. The ratios of lanosterol:14-desmethyllanosterol in the three strains indicate that the mutant transformed with erg11 showed more 14DM activity than wild-type yeast. The sterol analyses also indicated that the P. carinii 14DM can utilize the sterol substrates used by the S. cerevisiae 14DM and suggested that the yeast 14DM in the yeast cell utilizes 4α-methyl sterols better than the P. carinii enzyme.
authors.affiliation: Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221, USA.
authors.forename: Stephenson W
authors.initials: SW
authors.lastname: Nkinin
authors.forename: James R
authors.initials: JR
authors.lastname: Stringer
authors.forename: Scott P
authors.initials: SP
authors.lastname: Keely
authors.forename: Kenneth D R
authors.initials: KD
authors.lastname: Setchell
authors.forename: José-Luis
authors.initials: JL
authors.lastname: Giner
authors.forename: Edna S
authors.initials: ES
authors.lastname: Kaneshiro
mesh_terms: D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D055786:Gene Knockout Techniques; D007810:Lanosterol / Q000378:metabolism; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D045363:Pneumocystis carinii / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D058886:Sterol 14-Demethylase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013261:Sterols / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013261:Sterols; D007810:Lanosterol; D003577:Cytochrome P-450 Enzyme System; C526348:Erg11 protein, S cerevisiae; D058886:Sterol 14-Demethylase