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

Aouatef Bellamine, Anil T. Mangla, W. David Nes, Michael R. Waterman

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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 Δ(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 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.

Original languageEnglish
Pages (from-to)8937-8942
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number16
DOIs
StatePublished - Aug 3 1999

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