Structural and Biochemical Characterization of Mycobacterium tuberculosis CYP142

Abstract

The Mycobacterium tuberculosis cytochrome P450 enzyme CYP142 is encoded in a large gene cluster involved in metabolism of host cholesterol. CYP142 was expressed and purified as a soluble, low spin P450 hemoprotein. CYP142 binds tightly to cholesterol and its oxidized derivative cholest-4-en-3-one, with extensive shift of the heme iron to the high spin state. High affinity for azole antibiotics was demonstrated, highlighting their therapeutic potential. CYP142 catalyzes either 27-hydroxylation of cholesterol/cholest-4-en-3-one or generates 5-cholestenoic acid/cholest-4-en-3-one-27-oic acid from these substrates by successive sterol oxidations, with the catalytic outcome dependent on the redox partner system used. The CYP142 crystal structure was solved to 1.6 Å , revealing a similar active site organization to the cholesterol-metabolizing M. tuberculosis CYP125, but having a near-identical organization of distal pocket residues to the branched fatty acid oxidizing M. tuberculosis CYP124. The cholesterol oxidizing activity of CYP142 provides an explanation for previous findings that ⌬CYP125 strains of Mycobacterium bovis and M. bovis BCG cannot grow on cholesterol, because these strains have a defective CYP142 gene. CYP142 is revealed as a cholesterol 27-oxidase with likely roles in host response modulation and cholesterol metabolism. Tuberculosis (TB)3 is a debilitating and frequently fatal disease caused by the bacterium Mycobacterium tuberculosis. The disease usually presents as a pulmonary condition but can also affect other parts of the body (1). M. tuberculosis can also remain in a dormant phase in the host (latent TB), and a proportion of individuals with latent TB will later develop active TB (2). The emergence and proliferation of multidrugresistant and extensively drug-resistant M. tuberculosis strains have provided a major challenge for TB therapeutic development (3). Numerous M. tuberculosis clinical strains are now resistant to one or several front line and second line drugs, including antibiotics such as rifampicin, isoniazid, streptomycin, and ethambutol (4). TB and drug-resistant strains are prevalent in the less developed countries of Asia and Africa, and synergy between M. tuberculosis and HIV leads to high rates of TB infection and deaths in HIV-infected individuals (5). Consequently, there has been recent intensive research on the genetics and biochemistry of M. tuberculosis, as well as work to develop novel therapeutics and to identify new drug targets (6 -8). Recent successes include the development of 1,3-benzothiazin-4-ones that inhibit the decaprenylphosphoryl-␤-D-ribose 2Ј-epimerase enzyme and formation of cell wall arabinans, and clavulanate/␤-lactam combinations inhibiting formation of cell wall peptidoglycan (9, 10).The M. tuberculosis H37Rv and then the M. tuberculosis CDC1551 genome sequences provided key information on the M. tuberculosis proteome, highlighting the complexity of M. tuberculosis lipid metabolism as well as several genes previously found only in eukaryotes, e.g. the a...

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

“…Cholesterol degradation in Mtb is initiated by oxidation of a terminal methyl of the cholesterol side chain to a carboxylic acid by cytochrome P450 enzymes CYP125A1 (Rv3545c) and CYP142A1 (Rv3518c) (4,6,10). This oxidation reaction can occur with cholesterol itself, but does so more efficiently with cholest-4-en-3-one (4), the metabolite formed by oxidation of the 3-hydroxyl group to a ketone by a mycobacterial 3␤-hydroxysterol dehydrogenase (3␤-HSD, Rv1106c) (11)(12)(13).…”

Updating and curating metabolic pathways of TB

“…The side chain is first activated by conversion to the carboxylic acid by cytochrome P450 Cyp125 50, 51 . However, in some M. tuberculosis strains Cyp142 can compensate for loss of Cyp125 52 . The acyl-CoA synthetase responsible for the subsequent conversion to the CoA thioester has not been identified.…”

Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria

“…3-␤-hydroxysteroid dehydrogenases/isomerases and P450 cytochromes) in mycobacteria, and strains presenting deletions in these genes still retain their ability to grow on cholesterol using alternative enzymes (11,15,17,18,49). This redundancy, together with the detection of constitutive basal expression levels of these genes in the absence of cholesterol (18,20,22), ensures rapid production of the inducer, 3OChA, followed by complete transcription of the kstR regulon.…”

“…Although a compendium of biochemical and structural studies describe mycobacterial cholesterol catabolism, the degradation pathway and its transcriptional regulation are not yet fully established (5)(6)(7). The two first steps in cholesterol catabolism are the oxidation of C3 by cholesterol oxidases or 3-␤-hydroxysteroid dehydrogenase/ isomerases (8 -14) and the oxidation of C26 by P450 cytochromes (CYP125 and CYP142) (5,(15)(16)(17)(18), which are carried out sequentially and/or simultaneously. These biochemical reactions render the molecules cholest-4-en-3-ona (cholestenone) (after cholesterol C3 oxidation), 3-␤-hydroxy-5-cholestenoic acid (3OHChA) 3 (after cholesterol C26 oxidation), and 3-oxo-4-cholestenoic acid (3OChA) (after C3 and C26 oxidations) (Fig.…”