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Catabolism of the Cholesterol Side Chain in Mycobacterium tuberculosis Is Controlled by a Redox-Sensitive Thiol Switch.

ABSTRACT: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a highly successful human pathogen and has infected approximately one-third of the world's population. Multiple drug resistant (MDR) and extensively drug resistant (XDR) TB strains and coinfection with HIV have increased the challenges of successfully treating this disease pandemic. The metabolism of host cholesterol by Mtb is an important factor for both its virulence and pathogenesis. In Mtb, the cholesterol side chain is degraded through multiple cycles of ?-oxidation and FadA5 (Rv3546) catalyzes side chain thiolysis in the first two cycles. Moreover, FadA5 is important during the chronic stage of infection in a mouse model of Mtb infection. Here, we report the redox control of FadA5 catalytic activity that results from reversible disulfide bond formation between Cys59-Cys91 and Cys93-Cys377. Cys93 is the thiolytic nucleophile, and Cys377 is the general acid catalyst for cleavage of the ?-keto-acyl-CoA substrate. The disulfide bond formed between the two catalytic residues Cys93 and Cys377 blocks catalysis. The formation of the disulfide bonds is accompanied by a large domain swap at the FadA5 dimer interface that serves to bring Cys93 and Cys377 in close proximity for disulfide bond formation. The catalytic activity of FadA5 has a midpoint potential of -220 mV, which is close to the Mtb mycothiol potential in the activated macrophage. The redox profile of FadA5 suggests that FadA5 is fully active when Mtb resides in the unactivated macrophage to maximize flux into cholesterol catabolism. Upon activation of the macrophage, the oxidative shift in the mycothiol potential will decrease the thiolytic activity by 50%. Thus, the FadA5 midpoint potential is poised to rapidly restrict cholesterol side chain degradation in response to oxidative stress from the host macrophage environment.

SUBMITTER: Lu R

PROVIDER: S-EPMC5595149 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Catabolism of the Cholesterol Side Chain in Mycobacterium tuberculosis Is Controlled by a Redox-Sensitive Thiol Switch.

Lu Rui R Schaefer Christin M CM Nesbitt Natasha M NM Kuper Jochen J Kisker Caroline C Sampson Nicole S NS

ACS infectious diseases 20170816 9

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a highly successful human pathogen and has infected approximately one-third of the world's population. Multiple drug resistant (MDR) and extensively drug resistant (XDR) TB strains and coinfection with HIV have increased the challenges of successfully treating this disease pandemic. The metabolism of host cholesterol by Mtb is an important factor for both its virulence and pathogenesis. In Mtb, the cholesterol side ch ...[more]

PMID: 28786661

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Project description:The heteromeric acyl coenzyme A (acyl-CoA) dehydrogenase FadE28-FadE29 and the enoyl-CoA hydratase ChsH1-ChsH2, encoded by genes within the intracellular growth (igr) operon of Mycobacterium tuberculosis, catalyze the dehydrogenation of the cholesterol metabolite 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA), with a 3-carbon side chain, and subsequent hydration of the product 3-oxo-4,17-pregnadiene-20-carboxyl-CoA (3-OPDC-CoA) to form 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). The gene downstream of chsH2, i.e., ltp2, was expressed in recombinant Rhodococcus jostii RHA1 in combination with other genes within the igr operon. His-tagged Ltp2 copurified with untagged ChsH1-ChsH2, ChsH2, or the C-terminal domain of ChsH2, which contains a domain of unknown function (DUF35). Ltp2 in association with ChsH1-ChsH2 or just the DUF35 domain of ChsH2 was shown to catalyze the retroaldol cleavage of 17-HOPC-CoA to form androst-4-ene-3,17-dione and propionyl-CoA. Steady-state kinetic analysis using the Ltp2-DUF35 complex showed that the aldolase had optimal activity at pH 7.5, with a Km of 6.54 ± 0.90 ?M and a kcat of 159 ± 8.50 s-1 ChsH1-ChsH2 could hydrate only about 30% of 3-OPDC-CoA, but this unfavorable equilibrium could be overcome when the aldolase was present to remove the hydrated product, providing a rationale for the close association of the aldolase with the hydratase. Homologs of ChsH1, ChsH2, and Ltp2 are found in steroid-degrading Gram-positive and Gram-negative bacteria, suggesting that side chains of diverse steroids may be cleaved by aldolases in the bacteria.IMPORTANCE The C-C bond cleavage of the D-ring side chain of cholesterol was shown to be catalyzed by an aldolase. The aldolase associates with the hydratase that catalyzes the preceding reaction in the cholesterol side chain degradation pathway. These enzymes are encoded by genes within the intracellular growth (igr) operon of M. tuberculosis, and the operon was demonstrated previously to be linked to the pathogenicity and persistence of the bacteria in macrophages and in mice.

Project description:Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k(cat)/K(m) = 1000 +/- 100 M(-1) s(-1) versus 700 +/- 100 M(-1) s(-1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k(cat)/K(m) = 80 +/- 40 M(-1) s(-1)). In the presence of 3-HSA the K(m)(app) for O(2) was 100 +/- 10 microM. The crystal structure of HsaA to 2.5-A resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val(367)-Val(394)) could adopt two conformations differing by a rigid body rotation of 25 degrees around Arg(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.

Dataset Information

Catabolism of the Cholesterol Side Chain in Mycobacterium tuberculosis Is Controlled by a Redox-Sensitive Thiol Switch.

Publications

Catabolism of the Cholesterol Side Chain in Mycobacterium tuberculosis Is Controlled by a Redox-Sensitive Thiol Switch.

Similar Datasets

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