CYP123, a sterol C4-methyl oxidase, acts with CYP51 to initiate mycobacterial lanosterol catabolism

Abstract Mycobacteria exploit host cholesterol, both as a carbon source and as a source of catabolites proposed to modulate host immunity, but their capacity to catabolise other host sterols has not been established. Lanosterol, the first sterol intermediate of host cholesterol biosynthesis, accumulates in the activated macrophages these bacteria infect. Here we define the initiating steps of mycobacterial lanosterol catabolism, combining genomic enzymology, transcriptional profiling, mutagenesis, biochemistry, NMR and crystallography. We identify the cytochrome P450 CYP123 as a sterol C4-methyl oxidase. It is encoded with the lanosterol 14α-demethylase CYP51 in a co-oriented region conserved across most Mycobacteriaceae . The region carries no binding site for the cholesterol-responsive regulators KstR or KstR2, and in M. abscessus lanosterol induces both genes while cholesterol does not. CYP123 oxidises the C4α-methyl group of C14-demethylated sterols in three steps to the carboxylic acid, yet acts on neither lanosterol nor cholesterol, because Met82 excludes the C14-methylated precursor. An M82A variant binds and oxidises lanosterol. In a single reaction containing both recombinant M. smegmatis enzymes, CYP51 converts lanosterol to FF-MAS, which CYP123 then oxidises to the C4-carboxylic acid, so the two act in sequence. M. smegmatis , M. phlei and M. abscessus grow on lanosterol as a sole carbon source. Growth slows in M. smegmatis cyp123 and cyp51 mutants. Mycobacterial sterol catabolism therefore extends beyond cholesterol, and blocking the cholesterol route alone may not deprive these organisms of host sterols. Induction where lanosterol confers no growth advantage raises the possibility that the pathway also serves functions beyond carbon acquisition. Significance Mycobacteria feed on host cholesterol, a dependence long proposed as a drug target but never exploited therapeutically. This work shows that they also catabolise lanosterol, an early cholesterol precursor that accumulates in activated macrophages. Two cytochrome P450s encoded in a conserved, lanosterol-induced locus act in sequence: CYP51 removes the C14-methyl group, and CYP123 then oxidises the C4α-methyl group to a carboxylic acid. This explains why mycobacteria retain CYP51 despite being unable to make sterols, and identifies CYP123 as the first bacterial P450 sterol C4-methyl oxidase and a third enzymatic solution to C4-demethylation. The locus is conserved across more than 90% of Mycobacteriaceae , spanning pathogens of humans, livestock and the environment, and contributes to fitness during infection, making both enzymes attractive, azole-binding drug targets. Because cholesterol and lanosterol run through separate routes, depriving mycobacteria of host sterols would require blocking both.