Publications

• Cytochrome P450 125 (CYP125) catalyses C26-hydroxylation to initiate sterol side-chain degradation in Rhodococcus jostii RHA1.

  Rosloniec K.Z., Wilbrink M.H., Capyk J.K., Mohn W.W., Ostendorf M., van der Geize R., Dijkhuizen L., Eltis L.D.

  The cyp125 gene of Rhodococcus jostii RHA1 was previously found to be highly upregulated during growth on cholesterol and the orthologue in Mycobacterium tuberculosis (rv3545c) has been implicated in pathogenesis. Here we show that cyp125 is essential for R. jostii RHA1 to grow on 3-hydroxysterols ... >> More

  The cyp125 gene of Rhodococcus jostii RHA1 was previously found to be highly upregulated during growth on cholesterol and the orthologue in Mycobacterium tuberculosis (rv3545c) has been implicated in pathogenesis. Here we show that cyp125 is essential for R. jostii RHA1 to grow on 3-hydroxysterols such as cholesterol, but not on 3-oxo sterol derivatives, and that CYP125 performs an obligate first step in cholesterol degradation. The involvement of cyp125 in sterol side-chain degradation was confirmed by disrupting the homologous gene in Rhodococcus rhodochrous RG32, a strain that selectively degrades the cholesterol side-chain. The RG32 Omega cyp125 mutant failed to transform the side-chain of cholesterol, but degraded that of 5-cholestene-26-oic acid-3beta-ol, a cholesterol catabolite. Spectral analysis revealed that while purified ferric CYP125(RHA1) was < 10% in the low-spin state, cholesterol (K(D)(app) = 0.20 +/-0.08 microM), 5 alpha-cholestanol (K(D)(app) = 0.15 +/-0.03 microM) and 4-cholestene-3-one (K(D)(app) = 0.20 +/-0.03 microM) further reduced the low spin character of the haem iron consistent with substrate binding. Our data indicate that CYP125 is involved in steroid C26-carboxylic acid formation, catalysing the oxidation of C26 either to the corresponding carboxylic acid or to an intermediate state. << Less

  Mol. Microbiol. 74:1031-1043(2009) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Mycobacterial cytochrome p450 125 (cyp125) catalyzes the terminal hydroxylation of c27 steroids.

  Capyk J.K., Kalscheuer R., Stewart G.R., Liu J., Kwon H., Zhao R., Okamoto S., Jacobs W.R. Jr., Eltis L.D., Mohn W.W.

  Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinom ... >> More

  Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 +/- 0.02 microM and 0.27 +/-0.05 microm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9alpha-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis. << Less

  J. Biol. Chem. 284:35534-35542(2009) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest-4-en-3-one.

  Ouellet H., Guan S., Johnston J.B., Chow E.D., Kells P.M., Burlingame A.L., Cox J.S., Podust L.M., de Montellano P.R.

  The infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high-resolution mass spectrometric approaches. The a ... >> More

  The infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high-resolution mass spectrometric approaches. The analysis of lipid profiles from cells grown on cholesterol revealed that CYP125A1 is required to incorporate the cholesterol side-chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl-branched phthiocerol dimycocerosates. We observed that cholesterol-exposed cells lacking CYP125A1 accumulate cholest-4-en-3-one, suggesting that this is a physiological substrate for this enzyme. Reconstitution of enzymatic activity with spinach ferredoxin and ferredoxin reductase revealed that recombinant CYP125A1 indeed binds both cholest-4-en-3-one and cholesterol, efficiently hydroxylates both of them at C-27, and then further oxidizes 27-hydroxycholest-4-en-3-one to cholest-4-en-3-one-27-oic acid. We determined the X-ray structure of cholest-4-en-3-one-bound CYP125A1 at a resolution of 1.58 A. CYP125A1 is essential for growth of CDC1551 in media containing cholesterol or cholest-4-en-3-one. In its absence, the latter compound is toxic for both CDC1551 and H37Rv when added with glycerol as a second carbon source. CYP125A1 is a key enzyme in cholesterol metabolism and plays a crucial role in circumventing the deleterious effect of cholest-4-en-3-one. << Less

  Mol. Microbiol. 77:730-742(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.