Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis

• Published in: Biochemical and biophysical research communications 2003-02, Vol.301 (2), p.558-563
• Main Authors: Jackson, Colin J., Lamb, David C., Marczylo, Timothy H., Parker, Josie E., Manning, Nigel L., Kelly, Diane E., Kelly, Steven L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Azole; Cloning, Molecular; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - isolation & purification; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450; Genes, Bacterial; Humans; Lanosterol - metabolism; Lipids - chemistry; Molecular Sequence Data; Mycobacteria; Mycobacterium smegmatis - enzymology; Mycobacterium smegmatis - genetics; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - isolation & purification; Oxidoreductases - metabolism; Phylogeny; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Alignment; Sterol 14-Demethylase
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/S0006-291X(02)03078-4

The genetic locus encoding cytochrome P450 51 (CYP51; P450 14DM) in Mycobacterium smegmatis is described here together with confirmation of activity in lanosterol 14α-demethylation. The protein bound azole antifungals with high affinity and the rank order based on affinity matched the ranked order for microbiological sensitivity of the organism, thus supporting a possible role for CYP51 as a target in the antimycobacterial activity of these compounds. Non-saponifiable lipids were extracted from the bacteria grown on minimal medium. Unlike a previous report using growth on complex medium, no cholesterol was detected in two strains of M. smegmatis, but a novel lipid was detected. The genetic locus of CYP51 is discussed in relation to function; it is conserved as part of a putative operon in M. smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium bovis and consists of six open-reading frames including two CYPs and a ferredoxin under a putative Tet-R regulated promoter.