title: Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis.
abstract: Sterol 14alpha-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 14alpha-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 14alpha-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 14alpha-demethylases. It catalyzes 14alpha-demethylation of lanosterol, 24, 25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and (1)H NMR analysis. Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14alpha-methyl group and Delta(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 14alpha-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.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
authors.forename: A
authors.initials: A
authors.lastname: Bellamine
authors.forename: A T
authors.initials: AT
authors.lastname: Mangla
authors.forename: W D
authors.initials: WD
authors.lastname: Nes
authors.forename: M R
authors.initials: MR
authors.lastname: Waterman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D058886:Sterol 14-Demethylase; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase