Functional Redundancy of Steroid C26-monooxygenase Activity in Mycobacterium tuberculosis Revealed by Biochemical and Genetic Analyses

Abstract

One challenge to the development of new antitubercular drugs is the existence of multiple virulent strains that differ genetically. We and others have recently demonstrated that CYP125A1 is a steroid C 26 -monooxygenase that plays a key role in cholesterol catabolism in Mycobacterium tuberculosis CDC1551 but, unexpectedly, not in the M. tuberculosis H37Rv strain. This discrepancy suggests that the H37Rv strain possesses compensatory activities. Here, we examined the roles in cholesterol metabolism of two other cytochrome P450 enzymes, CYP124A1 and CYP142A1. In vitro analysis, including comparisons of the binding affinities and catalytic efficiencies, demonstrated that CYP142A1, but not CYP124A1, can support the growth of H37Rv cells on cholesterol in the absence of cyp125A1. All three enzymes can oxidize the sterol side chain to the carboxylic acid state by sequential oxidation to the alcohol, aldehyde, and acid. Interestingly, CYP125A1 generates oxidized sterols of the (25S)-26-hydroxy configuration, whereas the opposite 25R stereochemistry is obtained with CYP124A1 and CYP142A1. Western blot analysis indicated that CYP124A1 was not detectably expressed in either the H37Rv or CDC1551 strains, whereas CYP142A1 was found in H37Rv but not CDC1551. Genetic complementation of CDC1551 ⌬cyp125A1 cells with the cyp124A1 or cyp142A1 genes revealed that the latter can fully rescue the growth defect on cholesterol, whereas cells overexpressing CYP124A1 grow poorly and accumulate cholest-4-en-3-one. Our data clearly establish a functional redundancy in the essential C 26 -monooxygenase activity of M. tuberculosis and validate CYP125A1 and CYP142A1 as possible drug targets.Mycobacterium tuberculosis is the causative agent of human tubercular infection (tuberculosis) that, even today, poses a great threat to global human health. More than two billion people (a third of the world population) are infected latently with the bacterium, and of those individuals, ϳ10% will develop active tuberculosis infections during their lifetime. Currently, more than two million lives are claimed annually due to active M. tuberculosis infections (1). Among first world countries, the spread of M. tuberculosis has been kept mostly under control, but there has been a resurgence in developing countries in large part due to the emergence of multidrug-resistant bacterial strains that make the traditional frontline antibiotics less effective (2). Efforts continue on many fronts to understand this complex pathogen with a focus on identification of new drug targets.To proliferate within the macrophages, M. tuberculosis cells undergo a shift in metabolism from using carbohydrates to primarily utilizing host lipids (3-5). Sequencing of the M. tuberculosis genome revealed at least 250 genes predicted to be involved in lipid metabolism (6). A cholesterol catabolism cluster of 51 genes was recently identified in the genome of the M. tuberculosis-related actinomycete Rhodococcus jostti RHA1 (7). This region corresponds to the 82-gene cluster of M. tuberculos...

CYP63A2, a Catalytically Versatile Fungal P450 Monooxygenase Capable of Oxidizing Higher-Molecular-Weight Polycyclic Aromatic Hydrocarbons, Alkylphenols, and Alkanes

“…CYP102A1 (BM3) oxidizes fatty acids (C 12 to C 20 ) at internalchain carbons (-1, -2, and -3; 36:30:34) with no -hydroxylation (56). CYP125A1 from Rhodococcus jostii RAH1 (57) and CYP124, CYP125, and CYP142 from Mycobacterium tuberculosis -hydroxylate the sterol side chain of cholesterol and its 3-keto-4-ene derivative (58). Furthermore, CYP124 also -hydroxylates fatty acids and other long-chain lipids (59).…”

Pathway Profiling in Mycobacterium tuberculosis

“…source due to the compensatory enzymatic activity of Cyp142/ Rv3518c (24). The M. tuberculosis H37Rv ⌬igr mutant strain metabolizes C4 and C26 of cholesterol, indicating that the Cyp142/Rv3518c monooxygenase is functional in this mutant and that disruption of cholesterol degradation occurs after the C26 hydroxylation step (12).…”

Investigation of cytochromes P450 in myxobacteria: Excavation of cytochromes P450 from the genome of Sorangium cellulosum So ce56

“…PA genes are found in pathogenic and nonpathogenic mycobacterial species, actinomycetes, and some proteobacteria [68,69]. Moreover, CYP124E1 in Mycobacterium tuberculosis performed x-hydroxylation of methyl-branched lipids and degradation of the cholesterol [70,71]. CYP262A1 (sce2191)…”