Mycobacterium tuberculosis Utilizes a Unique Heterotetrameric Structure for Dehydrogenation of the Cholesterol Side Chain

Abstract

Compounding evidence supports the important role in pathogenesis that the metabolism of cholesterol by Mycobacterium tuberculosis (M. tuberculosis) plays. Elucidating the pathway by which cholesterol is catabolized is necessary to understand the molecular mechanism by which this pathway contributes to infection. Based on early metabolite identification studies in multiple actinomycetes, it has been proposed that cholesterol side chain metabolism requires one or more acyl-CoA dehydrogenases (ACADs). There are 35 genes annotated as encoding ACADs in the M. tuberculosis genome. Here we characterize a heteromeric ACAD encoded by Rv3544c and Rv3543c, formerly named fadE28 and fadE29, respectively. We now refer to genes Rv3544c and Rv3543c as chsE1 and chsE2 in recognition of their validated activity in cholesterol side chain dehydrogenation. Analytical ultracentrifugation and LC/UV experiments establish that ChsE1-ChsE2 forms an α2β2 heterotetramer, a new architecture for an ACAD. Our bioinformatic analysis and mutagenesis studies reveal that heterotetrameric ChsE1-ChsE2 has only two active sites. E241 in ChsE2 is required for catalysis of dehydrogenation by ChsE1-ChsE2. Steady state kinetic analysis establishes the enzyme is specific for an intact steroid ring system compared to hexahydroindanone substrates with specificity constants (kcat/KM) of 2.5 × 105 ± 0.5 s-1 M-1 vs 9.8 × 102 ± s-1 M-1 respectively, at pH 8.5. The characterization of a unique ACAD quaternary structure involved in sterol metabolism that is encoded by two distinct cistronic ACAD genes opens the way to identification of additional sterol metabolizing ACADs in M. tuberculosis and other actinomycetes through bioinformatic analysis.

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

“…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.…”

“…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.…”

“…M. tuberculosis cholesterol catabolism starts at the side chain while it is still connected to the steroid rings, thus, the requirement for the ␣ 2 ␤ 2 ACAD ChsE1/2 to catalyze the dehydrogenation reaction of the attached side chain (2). ChsE1/2 also has a preference for the four-ring steroid skeleton form over the two-ring form and does not have activity against short-chain substrates, indicating that the side chain is metabolized before degradation of the polycyclic skeleton (2). The current study shows that a second ␣ 2 ␤ 2 ACAD set (FadE26/27) catalyzes the dehydrogenation of 3␤-hydroxy-chol-5-en-24-oyl-CoA, indicating it is also involved in side chain degradation (1,2).…”

“…ChsE1/2 also has a preference for the four-ring steroid skeleton form over the two-ring form and does not have activity against short-chain substrates, indicating that the side chain is metabolized before degradation of the polycyclic skeleton (2). The current study shows that a second ␣ 2 ␤ 2 ACAD set (FadE26/27) catalyzes the dehydrogenation of 3␤-hydroxy-chol-5-en-24-oyl-CoA, indicating it is also involved in side chain degradation (1,2). Cholesterol catabolism appears to be primarily controlled by two transcriptional regulators, KstR and KstR2 (18).…”

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

“…strain Chol1 (10)(11)(12). Conversely, cholesterol catabolism has been extensively studied in Actinobacteria, especially M. tuberculosis and Rhodococcus species (13)(14)(15)(16)(17). During microbial degradation of steroids, the side chains are degraded via a process similar to the ␤-oxidation of fatty acids.…”