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1.
FIGURE 8.

FIGURE 8. From: Pathway Profiling in Mycobacterium tuberculosis.

Proposed function of igr operon. The function of the igr operon was assigned to be degradation of the 2′-propanoate side chain. The proposed catalytic function of each enzyme or enzyme complex is shown.

Suzanne T. Thomas, et al. J Biol Chem. 2011 December 23;286(51):43668-43678.
2.
FIGURE 2.

FIGURE 2. From: Pathway Profiling in Mycobacterium tuberculosis.

13C- or 14C-metabolic labeling of LDL-derived cholesterol. Isotopically labeled 2-[13C] or [14C]mevalonolactone is converted into mevalonate and is incorporated into the cholesterol biosynthesis pathway, resulting in labeled [1,7,5,22,26-13C]cholesterol or [1,7,5,22,26-14C]cholesterol. HMG-CoA, 3-hydroxy-3-methylglutarate coenzyme A.

Suzanne T. Thomas, et al. J Biol Chem. 2011 December 23;286(51):43668-43678.
3.
FIGURE 3.

FIGURE 3. From: Pathway Profiling in Mycobacterium tuberculosis.

Analysis of metabolically labeled LDL [13C]- or -[14C]cholesterol. A, shown is a TLC analysis of LDL [14C]cholesterol developed by charring (a) and autoradiography (b). B, shown is a MALDI-TOF MS spectrum of LDL [13C]cholesterol. Peaks labeled with asterisks are matrix ions. C, shown is a DEPT135 spectrum of isolated LDL [13C]cholesterol, confirming the position of isotopically labeled 13C carbons.

Suzanne T. Thomas, et al. J Biol Chem. 2011 December 23;286(51):43668-43678.
4.
FIGURE 5.

FIGURE 5. From: Pathway Profiling in Mycobacterium tuberculosis.

Structural characterization of Δigr metabolite 1. Structure of 1 was established from MS, tandem MS, and multidimensional NMR data. 1H and 13C NMR signals are listed. Dotted arrows represent HMBC correlations. Solid arrows represent COSY correlations. Only key correlations are displayed.

Suzanne T. Thomas, et al. J Biol Chem. 2011 December 23;286(51):43668-43678.
5.
FIGURE 6.

FIGURE 6. From: Pathway Profiling in Mycobacterium tuberculosis.

MS/MS/MS fragmentation analysis of Δigr metabolite 1. MS fragmentation data for the [MH-18]+ ion of metabolite 1 are shown. The loss of 18 Da is assumed to result from elimination of water from the enol tautomer. This fragmentation is not shown in the figure. A, [MH-18]+ = m/z 293 (natural abundance) is shown. B, [MH-18]+ = m/z 296 (three 13C labels) is shown. Sites of 13C labeling are indicated by asterisks.

Suzanne T. Thomas, et al. J Biol Chem. 2011 December 23;286(51):43668-43678.
6.
FIGURE 1.

FIGURE 1. From: Pathway Profiling in Mycobacterium tuberculosis.

M. tuberculosis cholesterol catabolic pathway. The enzymatic steps required for cholesterol metabolism are separated into steroid ring degradation (A) and side chain β-oxidation (B). Enzymes known to catalyze the depicted transformation are indicated in boldface. Catalytic steps for which the enzyme has not been identified are labeled with the predicted gene type.

Suzanne T. Thomas, et al. J Biol Chem. 2011 December 23;286(51):43668-43678.
7.
FIGURE 7.

FIGURE 7. From: Pathway Profiling in Mycobacterium tuberculosis.

Expression and purification of igr operon. A, constructs for expression of the igr operon in E. coli were prepared in vector pET28b. Each construct introduced an N-terminal His6 tag on the first gene product for purification. B, SDS-PAGE analysis of proteins purified by immobilized affinity chromatography from expression of constructs in A is shown. Protein identities were confirmed by trypsin digest and MALDI-TOF MS fingerprinting.

Suzanne T. Thomas, et al. J Biol Chem. 2011 December 23;286(51):43668-43678.
8.
FIGURE 4.

FIGURE 4. From: Pathway Profiling in Mycobacterium tuberculosis.

LC/MS analysis of M. tuberculosis H37Rv, Δigr, and igr complement extracts cultured with LDL [13C]cholesterol and natural abundance LDL cholesterol. A, total ion chromatograms of M. tuberculosis H37Rv, Δigr, and igr complement are shown. Only the total ion chromatograms from samples cultured with LDL [13C]cholesterol are shown. Major metabolites are labeled, with a full list in supplemental Table S1. Metabolite peaks A and B were only observed in M. tuberculosis H37Rv and igr complement, and peak C was only observed in Δigr. B, MS of M. tuberculosis H37Rv and igr complement cholesterol derived metabolite peaks A and B, which are ADD and AD, respectively. C, MS of cholesterol-derived metabolite C that accumulates in the Δigr mutant and is absent in the H37Rv and igr complemented strains is shown. The top panels of B and C are from growth on natural abundance LDL cholesterol, and the bottom panels are from growth on isotopically labeled LDL [13C]cholesterol.

Suzanne T. Thomas, et al. J Biol Chem. 2011 December 23;286(51):43668-43678.

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