FadA5 a Thiolase from Mycobacterium tuberculosis : A Steroid-Binding Pocket Reveals the Potential for Drug Development against Tuberculosis

Schaefer, C.M.; Lu, Rui; Nesbitt, Natasha M.; Schiebel, J.; Sampson, Nicole S.; Kisker, Caroline

doi:10.1016/j.str.2014.10.010

Cited by 41 publications

(55 citation statements)

References 44 publications

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“…In none of these instances was any CoA transferase activity detected. Finally, FadA6 shares 38% amino acid sequence identity with FadA5, a β-ketoacyl-CoA thiolase involved in cholesterol side-chain degradation (37) (Table 1), consistent with it catalyzing the thiolysis of a COCHEA-CoA ring-opened product containing a β-keto thioester moiety, to MOODA-CoA and acetyl-CoA.…”

Section: Resultsmentioning

confidence: 90%

Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria

Crowe

Casabon

Brown

et al. 2017

mBio

101

153

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Most mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene (ipdAB) and an acyl-CoA reductase gene (ipdC). In the presence of cholesterol, ΔipdC and ΔipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A ΔfadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by ΔipdF and ΔechA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a ΔipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis.

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Section: Resultsmentioning

confidence: 90%

Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria

Crowe

Casabon

Brown

et al. 2017

mBio

101

153

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“…Aerobic bacteria degrade steroid side chains through a series of reactions analogous to fatty acid ␤-oxidation. In M. tuberculosis, the thiolase FadA5 was proposed to catalyze the C-C bond cleavage of the side chain in the first two rounds of ␤-oxidation of the 8-carbon cholesterol side chain, releasing one molecule each of propionyl-CoA and acetyl-CoA and forming the steroid metabolite 3-OPC-CoA, with a 3-carbon side chain (11,12). In this study, we showed that cleavage of the 3-carbon side chain of this steroid metabolite during the last round of ␤-oxidation is catalyzed by an aldolase, involving Ltp2, instead of a thiolase.…”

Section: Discussionmentioning

confidence: 99%

Characterization of an Aldolase Involved in Cholesterol Side Chain Degradation in Mycobacterium tuberculosis

2018

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The heteromeric acyl coenzyme A (acyl-CoA) dehydrogenase FadE28-FadE29 and the enoyl-CoA hydratase ChsH1-ChsH2, encoded by genes within the intracellular growth () operon of , 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 , i.e.,, was expressed in recombinant RHA1 in combination with other genes within the 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 of 6.54 ± 0.90 μM and a of 159 ± 8.50 s 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. 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 () operon of , and the operon was demonstrated previously to be linked to the pathogenicity and persistence of the bacteria in macrophages and in mice.

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“…The region where the glutamine and α5 would be located in degradative thiolases is hypothesized to bind fatty acyl chains. In crystal structures of degradative thiolases from S. cerevisiae and A. thaliana (PDBs 1AFW and 2WU9) [26,27], 2-methyl-2,4-pentanediol and ethylene glycol are observed in this region, and in the crystal structure of a Mycobacterium tuberculosis degradative thiolase a steroid substrate mimic can be observed (PDB 4UBT) [28]. …”

Section: Discussionmentioning

confidence: 99%

Coenzyme A-free activity, crystal structure, and rational engineering of a promiscuous β-ketoacyl thiolase from Ralstonia eutropha

Fage

Keatinge‐Clay

2015

Journal of Molecular Catalysis B: Enzymatic

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Thiolases catalyze the formation of carbon-carbon bonds in diverse biosynthetic pathways. The promiscuous β-ketoacyl thiolase B of Ralstonia eutropha (ReBktB) has been utilized in the in vivo conversion of Coenzyme A (CoA)-linked precursors such as acetyl-CoA and glycolyl-CoA into β-hydroxy acids, including the pharmaceutically-important 3,4-dihydroxybutyric acid. Such thiolases could serve as powerful carbon-carbon bond-forming biocatalysts in vitro if handles less costly than CoA were employable. Here, thiolase activity is demonstrated toward substrates linked to the readily-available CoA mimic, N-acetylcysteamine (NAC). ReBktB was observed to catalyze the retro-Claisen condensation of several β-ketoacyl-S-NAC substrates, with a preference for 3-oxopentanoyl-S-NAC over 3-oxobutanoyl-, 3-oxohexanoyl-, and 3-oxoheptanoyl-S-NAC. A 2.0 Å-resolution crystal structure, in which the asymmetric unit consists of four ReBktB tetramers, provides insight into acyl group specificity and how it may be engineered. By replacing an active site methionine with an alanine, a mutant possessing significant activity towards α-methyl substituted, NAC-linked substrates was engineered. The ability of ReBktB and its engineered mutants to utilize NAC-linked substrates will facilitate the in vitro biocatalytic synthesis of diketide chiral building blocks from feedstock molecules such as acetate and propionate.

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FadA5 a Thiolase from Mycobacterium tuberculosis : A Steroid-Binding Pocket Reveals the Potential for Drug Development against Tuberculosis

Cited by 41 publications

References 44 publications

Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria

Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria

Characterization of an Aldolase Involved in Cholesterol Side Chain Degradation in Mycobacterium tuberculosis

Coenzyme A-free activity, crystal structure, and rational engineering of a promiscuous β-ketoacyl thiolase from Ralstonia eutropha

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