Lanosterol

About: Lanosterol is a research topic. Over the lifetime, 1239 publications have been published within this topic receiving 36737 citations. The topic is also known as: (3β)-lanosta-8,24-dien-3-ol & (3β,20R)-lanosta-8,24-dien-3-ol.

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

Abstract: Sterol 14α-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 14α-demethylases might be present in this bacterium. Using genomic DNA from mycobacterial strain H37Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14α-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 SDS/PAGE, and binds both sterol substrates and azole inhibitors of P450 14α-demethylases. It catalyzes 14α-demethylation of lanosterol, 24,25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and 1H NMR analysis. Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14α-methyl group and Δ8(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 14α-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.

A gain-of-function mutation in the transcription factor Upc2p causes upregulation of ergosterol biosynthesis genes and increased fluconazole resistance in a clinical Candida albicans isolate.

Abstract: In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate the expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Overexpression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Overexpression of ERG11 leads to the increased production of lanosterol demethylase, which contributes to azole resistance in clinical isolates of C. albicans, but the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately upregulated with ERG11 in a fluconazole-resistant clinical isolate compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single-nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug-susceptible strain resulted in constitutive upregulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole-resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to the increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans.