Sterol 14␣-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 14␣-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 14␣-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 SDS͞PAGE, and binds both sterol substrates and azole inhibitors of P450 14␣-demethylases. It catalyzes 14␣-demethylation of lanosterol, 24,25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC͞MS and 1 H NMR analysis. Both f lavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14␣-methyl group and ⌬ 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 14␣-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.

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Nuclear Overhauser difference spectroscopy and variable temperature studies of the 9.3,19-cyclopropyl sterols 24,25-dehydropollinastanol (4,4-desmethyl-5a-cycloart-24-en-3fi-ol) and cyclolaudenol [(24S)-24-methyl-5a-cycloart-25(27)-en-3,3-ol] have shown the solution conformation ofthe B/C rings to be twist-chair/twist-boat rather than boat/ chair as suggested in the literature. This is very similar to the known crystal structure conformation of 9,3,19-cyclopropyl sterols. The effect of these conformations on the molecular shape is highly significant: the first conformation orients into a pseudoplanar or flat shape analogous to lanosterol, whereas the latter conformation exhibits a bent shape. The results are interpreted to imply that, for conformational reasons, cyclopropyl sterols can be expected to maintain the pseudoplanar shape in membrane bilayers.For reasons that are still unknown, organisms having a photosynthetic lineage cyclize squalene oxide to the 9f3,19-cyclopropyl sterol (CS) cycloartenol, while organisms having an evolutionary history that is completely nonphotosynthetic cyclize squalene oxide to the isomeric tetracycle lanosterol (1, 2). Cycloartenol has been shown to isomerize to lanosterol under acidic conditions (3) and during routine metabolism in plants (2). In lanosterol the 8,9 double bond approximates a trans structure for the B/C ring junction, maintaining a flat conformation of the molecule. However in CSs the 9,10 cyclopropyl bridgehead and the ,B oriented hydrogen at C-8 approximate a syn-cis configuration at the A/C and B/C ringjunctions. Some investigators believe (3-8) that this configurational disposition bends the plane of the molecule at the B/C ring junction through almost 90°. Dreiding models also show the molecule is no longer flat in the bent shape but neither they nor stereochemical considerations of polycyclic systems show that CSs could orient into the exaggerated "curvilinear belt" described by Bloch (4). That the configuration of the ring junctures influences the overall conformation of the molecule is known (8) in the isomeric pair of saturated sterols cholestanol and coprostanol. Here, inversion of the configuration of H-5 changes the A/B ring juncture stereochemistry from flat

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