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

Compounding evidence supports the important role in pathogenesis that the metabolism of cholesterol by Mycobacterium tuberculosis plays. Elucidating the pathway by which cholesterol is catabolized is necessary to understand the molecular mechanism by which this pathway contributes to infection. On t...

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Bibliographic Details
Published in:Biochemistry (Easton) 2013-04, Vol.52 (17), p.2895-2904
Main Authors: Thomas, Suzanne T, Sampson, Nicole S
Format: Article
Language:English
Subjects:
Acyl-CoA Dehydrogenase - metabolism
Biophysics
Biopolymers - metabolism
Cholesterol - metabolism
Chromatography, Gel
Hydrogenation
Mycobacterium tuberculosis
Mycobacterium tuberculosis - enzymology
Mycobacterium tuberculosis - metabolism
Nuclear Magnetic Resonance, Biomolecular
Plasmids
Spectrophotometry, Ultraviolet
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Summary:Compounding evidence supports the important role in pathogenesis that the metabolism of cholesterol by Mycobacterium tuberculosis plays. Elucidating the pathway by which cholesterol is catabolized is necessary to understand the molecular mechanism by which this pathway contributes to infection. On the basis of 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, respectively, in recognition of their validated activity in cholesterol side chain dehydrogenation. Analytical ultracentrifugation and liquid chromatography–ultraviolet 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 versus hexahydroindanone substrates with specificity constants (k cat/K M) of (2.5 ± 0.5) × 105 s–1 M–1 versus 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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi4002979