Mycobacterium tuberculosis Cholesterol Catabolism Requires a New Class of Acyl Coenzyme A Dehydrogenase

In this month’s issue of the Journal of Bacteriology, Wipperman et al. establish a new category of acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) that form unique 2 2 heterotetrameric complexes required to catalyze unsaturation reactions in -oxidation of steroid-CoA substrates (1). The described 2 2 ACADs allow Mycobacterium tuberculosis, and perhaps other pathogens, to take advantage of ubiquitous cholesterol as a readily available carbon source. The M. tuberculosis genome contains six sets of ACAD genes (fadE genes) that are regulated by cholesterol, with each set of genes found adjacent to each other within the same operon (1). The current study provides compelling evidence that all of these adjacent cholesterol-regulated genes encode members of a new 2 2 heterotetrameric ACAD class. The Sampson laboratory previously demonstrated that two of these genes, chsE1 and chsE2, encode an 2 2 ACAD required for the dehydrogenation of the cholesterol side chain (2). The current study extends the initial characterization of ChsE1/2 to five additional sets of 2 2 ACADs. Of the six 2 2 ACAD sets, five are induced by cholesterol and likely important for cholesterol catabolism, while one set is repressed by cholesterol and is not likely involved in cholesterol catabolism. Acyl-CoA dehydrogenases were first characterized in the 1950s by Beinert and colleagues as enzymes that catalyze the unsaturation of acyl-CoA thioesters in -oxidation (3, 3a). Prior to the recent studies by the Sampson laboratory, ACADs had only been characterized that catalyze the unsaturation of short-, medium-, and long-chain acyl-CoA thioesters, and all were homotetrameric or homodimeric in structure (4). Thus, the recent report on ChsE1/E2 was the first characterization of an 2 2 heterotetrameric ACAD (2). Homotetrameric ACADs have four active sites with four flavin adenine dinucleotide (FAD) cofactors. In five of the six 2 2 ACADs, only one of the adjacent ACAD subunits contains the catalytic residue required for deprotonation of the acyl-CoA substrate. The authors demonstrate that the 2 2 ACADs have one active site and one FAD per heterodimer instead of two. Five of the six genome adjacent fadE pairs formed only heteromeric complexes when expressed in vitro, while the sixth set, FadE17/18, did not form soluble protein. A control pair of fadE genes that are transcribed in the same operon, but are not adjacent pairs, formed typical homomeric complexes only when expressed in vitro and did not have characteristics of the 2 2 ACAD class. Thus, the 2 2 ACADs are formed from two heterodimers in which each specific monomer is required to form a unique active site. The M. tuberculosis genome contains a disproportionally large number of lipid and fatty acid metabolic genes including 35 members of the fadE family (5). This apparent redundancy in the genome has long been a mystery. The study by Wipperman et al. has unraveled a piece of this mystery by establishing a role for 11 fadE genes that encode six highly specific ACAD complexes. It has also become clear that in vivo, M. tuberculosis prefers to consume fatty acids and lipids; thus, it has preserved the ability to metabolize complex lipids such as cholesterol. Mutants with defects in the glyoxylate, methylcitrate, and gluconeogenic pathways, important for evenand odd-chain fatty acid metabolism, are all severely attenuated in mouse models of tuberculosis (TB) infection (6–9). M. tuberculosis isolated from murine lungs readily metabolize fatty acids but not carbohydrates (10). A variety of lipids are available to M. tuberculosis during different stages of infection, including cholesterol, an abundant and essential component of animal cell membranes. Catabolism of cholesterol provides a carbon source for energy production and M. tuberculosis lipid synthesis (11). Cholesterol is broken down to acetyl-CoA for the tricarboxylic acid (TCA) cycle, propionyl-CoA for the methylcitrate cycle or lipid synthesis, and pyruvate for the generation of acetyl-CoA, or potentially to drive gluconeogenesis. Studies with mice demonstrate that high dietary cholesterol enhances TB infection (12). The mere fact that M. tuberculosis, an obligate human pathogen, has maintained over 80 genes involved in cholesterol metabolism points to the importance of cholesterol to the pathogen. In vivo experiments have produced mixed results in demonstrating an essential role for cholesterol metabolism. Experiments with strains with deletions of genes coding for specific parts of the cholesterol degradative pathway exhibited attenuated phenotypes during infection of mice and guinea pigs that were attributed to accumulation of toxic intermediates (13–15). Deletion of the mce4 cholesterol transport locus resulted in decreased growth on cholesterol and decreased virulence in activated macrophages and murine infection (16), supporting an important role for cholesterol catabolism in M. tuberculosis pathogenesis. However, deletion of M. tuberculosis Rv1106c, which encodes the first step in cholesterol catabolism and is necessary for in vitro growth on cholesterol, did not result in attenuation in macrophage or guinea pig infections (17), indicating that cholesterol catabolism may not be essential for infection. Nevertheless, the buildup of toxic intermediates upon interruption of cholesterol degradation and the resulting bacteriostasis or bacillus death make the catabolic enzymes of cholesterol degradation attractive drug targets (18). Cholesterol is made up of an 8-carbon side chain and a polycyclic steroid ring moiety with A, B, and C cyclohexane rings and a cyclopentane D ring. M. tuberculosis cholesterol catabolism