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

Sterol 14 alpha-demethylase encoded by CYP51 is a mixed-function oxidase in volved in sterol synthesis in eukaryotic organisms. Completion of the Mycob acterium tuberculosis genome project revealed that a protein having homolog y to mammalian 14 alpha-demethylases might be present in this bacterium. Us ing genomic DNA. from mycobacterial strain H(37)Rv, we have established una mbiguously that the CYP51-like gene encodes a bacterial sterol 14 alpha-dem ethylase. Expression of the M. tuberculosis CYP51 gene in Escherichia coli yields a P450, which, when purified to homogeneity, has the predicted molec ular mass, ca. 50 kDa on SDS/PAGE, and binds both sterol substrates and azo le inhibitors of P450 14 alpha-demethylases. It catalyzes 14 alpha-demethyl ation of lanosterol, 24,25-dihydrolanosterol, and obtusifoliol to produce t he 8,14-dienes stereoselectively as shown by GC/MS and H-1 NMR analysis, Bo th flavodoxin and ferredoxin redox systems are able to support this enzymat ic activity. Structural requirements of a 14 alpha-methyl group and Delta(8 (9))-bond were established by comparing binding of pairs of sterol substrat e that differed in a single molecular feature, e.g., cycloartenol paired wi th lanosterol. These substrate requirements are similar to those establishe d for plant and animal P450 14 alpha-demethylases. From the combination of results, the interrelationships of substrate functional groups within the a ctive site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.