Unraveling Cholesterol Catabolism in Mycobacterium tuberculosis: ChsE4-ChsE5 α₂β₂ Acyl-CoA Dehydrogenase Initiates β-Oxidation of 3-Oxo-cholest-4-en-26-oyl CoA

Abstract

The metabolism of host cholesterol by Mycobacterium tuberculosis (Mtb) is an important factor for both its virulence and pathogenesis, although how and why cholesterol metabolism is required is not fully understood. Mtb uses a unique set of catabolic enzymes that are homologous to those required for classical β-oxidation of fatty acids but are specific for steroid-derived substrates. Here, we identify and assign the substrate specificities of two of these enzymes, ChsE4-ChsE5 (Rv3504-Rv3505) and ChsE3 (Rv3573c), that carry out cholesterol side chain oxidation in Mtb. Steady-state assays demonstrate that ChsE4-ChsE5 preferentially catalyzes the oxidation of 3-oxo-cholest-4-en-26-oyl CoA in the first cycle of cholesterol side chain β-oxidation that ultimately yields propionyl-CoA, whereas ChsE3 specifically catalyzes the oxidation of 3-oxo-chol-4-en-24-oyl CoA in the second cycle of β-oxidation that generates acetyl-CoA. However, ChsE4-ChsE5 can catalyze the oxidation of 3-oxo-chol-4-en-24-oyl CoA as well as 3-oxo-4-pregnene-20-carboxyl-CoA. The functional redundancy of ChsE4-ChsE5 explains the in vivo phenotype of the igr knockout strain of Mycobacterium tuberculosis; the loss of ChsE1-ChsE2 can be compensated for by ChsE4-ChsE5 during the chronic phase of infection. The X-ray crystallographic structure of ChsE4-ChsE5 was determined to a resolution of 2.0 Å and represents the first high-resolution structure of a heterotetrameric acyl-CoA dehydrogenase (ACAD). Unlike typical homotetrameric ACADs that bind four flavin adenine dinucleotide (FAD) cofactors, ChsE4-ChsE5 binds one FAD at each dimer interface, resulting in only two substrate-binding sites rather than the classical four active sites. A comparison of the ChsE4-ChsE5 substrate-binding site to those of known mammalian ACADs reveals an enlarged binding cavity that accommodates steroid substrates and highlights novel prospects for designing inhibitors against the committed β-oxidation step in the first cycle of cholesterol side chain degradation by Mtb.

The Minimal Unit of Infection:Mycobacterium tuberculosisin the Macrophage

“…Consistent with the idea that lipids are important nutrients the early genome annotation efforts revealed that M. tuberculosis has an expanded repertoire of genes associated with lipid metabolism and β-oxidation, which exceeded 250 genes (57). Although several of the M. tuberculosis genes originally annotated as having a β-oxidation functions are now known to have anabolic activities (58, 59) or act in multimeric complexes to oxidize lipids (60, 61) the M. tuberculosis genome still encodes more genes involved in lipid metabolism and β-oxidation relative to other bacteria. Another critical pathway that is required when bacteria metabolize lipids is the anaplerotic glyoxylate cycle and a key enzyme in this pathway is isocitrate lyase (Icl).…”

Heterologous expression and characterization of a 3-ketosteroid-∆1-dehydrogenase from Gordonia neofelifaecis and its utilization in the bioconversion of androst-4,9(11)-dien-3,17-dione

“…Microbial transformation could be carried out under mild reaction conditions with excellent yields of products and remarkable regio- and stereo-selectivity, which is hardly available for chemical synthesis. Therefore, for producing novel steroidal drugs and generating active pharmaceutical ingredients, microbial transformation is employed as a novel, efficient and economical tool (Donova 2007; García et al 2012; Yang et al 2015). For example, side chains of phytosterol, a byproduct from soybeans, sugar and paper industries, can be selectively degraded by a process similar to the β-oxidation of fatty acids, yielding 17-ketosteroids (Wei et al 2010).…”

“…The efficiency of enzymatic processes and purity of their products have obvious advantages in comparison with multi-steps chemical syntheses of hormonal drugs. However, the development of steroid biotechnology requires further studies of microorganisms able to degrade/modify steroids as well as enzymes catalyzing these reactions on the molecular level (Yang et al 2015). …”

α-Methyl Acyl CoA Racemase Provides Mycobacterium tuberculosis Catabolic Access to Cholesterol Esters

“…FadD19 is an essential enzyme when Mtb is grown on cholesterol 16 and is the only fatty CoA ligase that has been identified to esterify the terminal cholesterol carboxylic acid. However, FadD19 is not stereoselective as it accepts both the 25 R and 25 S carboxylic acids 6 (Scheme 1). Thus, the metabolic pathway that Mtb utilizes to activate cholesterol to its CoA ester provides both the 25 R or 25 S diastereomers of 3-oxo-cholest-4-en-26-oyl CoA (3-OCS-CoA).…”

“…5, 6, 8, 9 ChsE4-ChsE5 is the only ACAD protein regulated by KstR1 that can oxidize 3-OCS-CoA the first acyl-CoA metabolite in the side chain β-oxidation cycle. 6 …”