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

Abstract

SummaryThe 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 highresolution 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 Å. 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.

Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis

“…Both the in vitro nature of experiments exploring this pathway and a level of facultative promiscuity suggested by current data for some of the biochemically investigated enzymes contribute to the limitations inherent in our understanding of the pathway. Thus, it is established that 3␤-hydroxysteroid dehydrogenase (3␤-HSD) but not KstD can transform cholesterol before Cyp125 catalyzes the first side chain reaction, and that Cyp125 is capable of transforming cholesterol or 4-cholesten-3-one (21,22,36). Likewise, side chain degradation can proceed to its conclusion before the action of either KstD or KshA (11), but both of these enzymes can transform intermediates of side chain degradation.…”

“…Interestingly, the order of particular reactions seems to be species-dependent. For example, 3␤-hydroxy-steroid dehydrogenase (3␤-HSD) of Mtb CDC1551 and Mycobacterium bovis BCG are able to transform cholesterol (21,22), whereas the corresponding ring-degrading enzyme of R. jostii RHA1 requires the activity of the first side chain-transforming enzyme, 26-cholesterol hydroxylase (Cyp125) (23).…”

Cholesterol Analogs with Degradation-resistant Alkyl Side Chains Are Effective Mycobacterium tuberculosis Growth Inhibitors

“…The ␤-oxidation of the side chain gives rise to 4-androstenedione, which can undergo further degradation (3). The acetyl-and propionoyl-CoA fragments obtained from the side chain are incorporated into cellular lipids and virulence factors (14,15).…”

“…Mtb without functional CYP125A1 and CYP142A1 is unable to grow on cholesterol as a carbon source, but grows readily on carbon sources such as glycerol, glucose, or acetate (6,14). In these mycobacteria, cholesterol accumulates in the form of cholest-4-en-3-one (14).…”

Assessing the progress of Mycobacterium tuberculosis H37Rv structural genomics

“…The solubility of secreted target proteins may be improved by co-expressing the soluble domain of the membrane-anchored disulphide bond-forming (Dsb) protein (Rv2969c) (51,52). Similarly, GlgB (Rv1326c), Cyp125A1 (Rv3545c), and tuberculosinyl transferase (Rv3378c) were converted to soluble proteins by co-producing E. coli chaperone proteins (17,53,54).…”