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 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 α(2)β(2) 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) × 10(5) s(-1) M(-1) versus 9.8 × 10(2) 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.

Figure 1. UV-visible spectra of purified ChsE proteins

Purified ChsE2 (50 μM), ChsE1-ChsE2 (15 μM), and ChsE1-ChsE2_E241Q (15 μM). Relative spectral maxima at 370 nm and 446 nm for ChsE1-ChsE2 and ChsE1-ChsE2_E241Q are characteristic of oxidized flavin. No flavin absorbances were observed for ChsE2 alone.

Figure 2. Analytical gel filtration of ChsE2 and ChsE1-ChsE2 complexes

ChsE2 expressed in E. coli and M. smegmatis and ChsE1-ChsE2 and ChsE1-ChsE2_E241Q expressed in E. coli were analyzed by analytical gel filtration on a Superdex 75 column under identical conditions. Signal at 220 nm was normalized to 1.0. The ChsE1-ChsE2 protein formed a stable complex with a higher molecular weight than ChsE2 expressed alone in E. coli or M. smegmatis.

Figure 3. Analytical ultracentrifugation sedimentation equilibrium data for ChsE proteins

(A) ChsE2 and (B) ChsE1-ChsE2 were analyzed at three concentrations ranging from 1 μM to 11 μM at centrifugation speeds of 20k, 25k, and 30k at 20 °C. A representative fit for each sample is shown. The solid line shows the fit of the data to the ideal species model and the residuals of the fit are graphed below. The global fit for each protein provided molecular weights of 42.0 ± 0.5 kDa and 156 ± 1 kDa for ChsE2 and ChsE1-ChsE2, respectively.

Figure 4. Reverse phase LC/UV chromatogram of ChsE1-ChsE2

Peak a and peak b were identified as ChsE2 and ChsE1, respectively, by deconvolution of multiple charged states in the corresponding ESI+ MS spectra. The absorbance peaks were integrated and relative concentrations were determined from the calculated extinction coefficients of ChsE1 and ChsE2, ε₂₈₀(ChsE1)= 35,410 M^-1 cm^-1; ε₂₈₀ (ChsE2)=58,900 M^-1cm^-1.

Figure 5. Bioinformatic analysis of ChsE1 and ChsE2

(A) Sequence alignment of ChsE1 and ChsE2 against human and bacterial ACADS; MCAD (P11310), SCAD (P16219), LCAD (P28330), SBCAD (P45954), iBD (Q9UKU7), IVD (P26440), GD (Q92947), VLCAD (P49748), ACAD9 (Q9H845), ACAD10 (Q6JQN1), ACAD11 (Q709F0), 1BUC (SCAD Megasphaera elsdenii, Q06319), 3NF4 (Mycobacterium thermoresistibile, G7CDN2). Residues in green bind the isoalloxazine and ribityl diphosphate moieties of FAD, residues in blue bind adenosine of FAD, residues in purple bind CoA, and residues in yellow are the catalytic base. (B) Homology model of FAD binding residues and (C) octanoyl-CoA binding residues in ChsE1-ChsE2 based on human MCAD homodimer (PDB 1EGC). FAD is displayed in yellow and octanoyl-CoA is displayed in green. FAD and CoA cofactors are shown adjacent to conserved binding residues.

Figure 6. Characterization of dehydrogenated product by ChsE1-ChsE2

¹H spectra (700 MHz) of substrate 1 and ChsE1-ChsE2 assay product 3 illustrating the changes in the methyl (B) and alkene (C) regions. The alkene stereochemistry is represented as (E), but was not determined.

Figure 7. Optimization of dehydrogenase activity of ChsE1-ChsE2

(A) 3-oxo-4-pregnene-20-carboxyl-CoA 1 and 1β- (2’-propanoyl-CoA)-3aα-H-7aβ-methylhexahydro-4-indanone 2, substrates of ChsE1-ChsE2. (B) pH and (C) ionic strength optimization of dehydrogenase activity. Assays were conducted with 50 μM of substrate 1 and 250 μM ferricenium hexafluorophosphate at 25 °C. Assays in C were conducted in 100 mM TAPS buffer pH 8.5.

Scheme 1

An igr disrupted strain of M. tuberculosis accumulates methyl 1β-(2’-propanoate)-3aα-H-4α-(3’-propanoic acid)-7aβ-methylhexahydro- 5-indanone when cultured with cholesterol.