Post-translational Succinylation of Mycobacterium tuberculosis Enoyl-CoA Hydratase EchA19 Slows Catalytic Hydration of Cholesterol Catabolite 3-Oxo-chol-4,22-diene-24-oyl-CoA

Bonds, Amber C.; Yuan, Tianao; Werman, Joshua M.; Jang, Jungwon; Lu, Rui; Nesbitt, Natasha M.; García‐Díaz, Miguel; Sampson, Nicole S.

doi:10.1021/acsinfecdis.0c00329

Cited by 18 publications

(19 citation statements)

References 53 publications

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“…We previously reported that EchA19 is an enoyl-CoA hydratase that is regulated by the KstR1 repressor and has been implicated in the cholesterol side-chain catabolism in Mtb, where it preferentially catalyzes the hydration of 3-OCDO-CoA among other sterol enoyl-CoA esters. 17 EchA19, structurally unlike ChsH3, possesses a canonical crotonase-like structure and is a member of the crotonase (PRK-07799) superfamily, which is known to catalyze the hydration of enoyl-CoAs with an opposite stereochemistry to the MaoC-like family.…”

Section: Resultsmentioning

confidence: 99%

“…3-Oxo-pregna-4,17-diene-20-carboxyl-CoA (3-OPDC-CoA), 3-OCDO-CoA, 3-OCDS-CoA, and trans -2-octenoyl-CoA were synthesized as previously reported. 17 To make (22 R )-HOCO-CoA or (3 R )-hydroxyoctanoyl-CoA, 200 μM 3-OCDO-CoA or trans -2-octenoyl-CoA was incubated with 1 μM ChsH3 in buffer D in a 10 mL reaction volume at 25 °C for 2 h. The reaction was quenched by adding 100 μL of TFA and product was purified by HPLC on a C18 column using 10 mM ammonium acetate with a linear gradient of acetonitrile from 5 to 100% over 40 min. (22 S )-HOCO-CoA was synthesized in a similar manner by using EchA19.…”

Section: Methodsmentioning

confidence: 99%

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Enzymatic β-Oxidation of the Cholesterol Side Chain in Mycobacterium tuberculosis Bifurcates Stereospecifically at Hydration of 3-Oxo-cholest-4,22-dien-24-oyl-CoA

et al. 2021

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The unique ability of Mycobacterium tuberculosis (Mtb) to utilize host lipids such as cholesterol for survival, persistence, and virulence has made the metabolic pathway of cholesterol an area of great interest for therapeutics development. Herein, we identify and characterize two genes from the Cho-region (genomic locus responsible for cholesterol catabolism) of the Mtb genome, chsH3 (Rv3538) and chsB1 (Rv3502c). Their protein products catalyze two sequential stereospecific hydration and dehydrogenation steps in the β-oxidation of the cholesterol side chain. ChsH3 favors the 22 S hydration of 3-oxo-cholest-4,22-dien-24-oyl-CoA in contrast to the previously reported EchA19 (Rv3516), which catalyzes formation of the (22 R )-hydroxy-3-oxo-cholest-4-en-24-oyl-CoA from the same enoyl-CoA substrate. ChsB1 is stereospecific and catalyzes dehydrogenation of the ChsH3 product but not the EchA19 product. The X-ray crystallographic structure of the ChsB1 apo-protein was determined at a resolution of 2.03 Å, and the holo-enzyme with bound NAD + cofactor was determined at a resolution of 2.21 Å. The homodimeric structure is representative of a classical NAD + -utilizing short-chain type alcohol dehydrogenase/reductase, including a Rossmann-fold motif, but exhibits a unique substrate binding site architecture that is of greater length and width than its homologous counterparts, likely to accommodate the bulky steroid substrate. Intriguingly, Mtb utilizes hydratases from the MaoC-like family in sterol side-chain catabolism in contrast to fatty acid β-oxidation in other species that utilize the evolutionarily distinct crotonase family of hydratases.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Enzymatic β-Oxidation of the Cholesterol Side Chain in Mycobacterium tuberculosis Bifurcates Stereospecifically at Hydration of 3-Oxo-cholest-4,22-dien-24-oyl-CoA

et al. 2021

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“…We previously reported that EchA19 is an enoyl-CoA hydratase that is regulated by the KstR1 repressor and has been implicated in the cholesterol side chain catabolism in Mtb, where it preferentially catalyzes the hydration of 3-OCDO-CoA among other sterol enoyl-CoA esters. 16 EchA19, structurally unlike ChsH3, possesses a canonical crotonase-like structure and is a member of the crotonase (PRK-07799) superfamily, which is known to catalyze the hydration of enoyl-CoAs with an opposite stereochemistry to the MaoC-like family.…”

Section: Resultsmentioning

confidence: 99%

Enzymatic β-Oxidation of the Cholesterol Side Chain in Mycobacterium Tuberculosis Bifurcates Stereospecifically at Hydration of 3-Oxo-Cholest-4,22- Dien-24-Oyl-CoA

Yuan

Werman

Yin

et al. 2021

Preprint

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The unique ability of Mycobacterium tuberculosis (Mtb) to utilize host lipids such as cholesterol for survival, persistence, and virulence has made the metabolic pathway of cholesterol an area of great interest for therapeutics development, and bioproduction of valuable sterol intermediates. Herein, we identify and characterize two genes from the <a></a><a>Cho-region of the Mtb genome</a>, chsH3 (Rv3538) and chsB1 (Rv3502c). Their protein products catalyze <a></a><a>two sequential stereospecific</a>hydration and dehydrogenation steps in the b-oxidation of the cholesterol side chain. ChsH3 favors the 22S hydration of 3-oxo-cholest-4,22-dien-24-oyl-CoA in contrast to the previously reported EchA19 (Rv3516) which catalyzes formation of the (22R)-hydroxy-3-oxo-cholest-4-en-24-oyl-CoA from the same enoyl-CoA substrate. ChsB1 is stereospecific and catalyzes dehydrogenation of the ChsH3 product, but not the EchA19 product. The X-ray crystallographic structure of the ChsB1 apo-protein was determined at a resolution of 2.03 Å and the holo-enzyme with bound NAD+ cofactor at 2.21 Å.The homodimeric structure is representative of a classical NAD+ utilizing short-chain type alcohol dehydrogenase/reductase, including a Rossmann-fold motif, but exhibits a unique substrate binding site architecture that is of greater length and width than its homologous counterparts, likely to accommodate the bulky steroid substrate. Intriguingly, Mtb utilizes MaoC-like hydratases in sterol side-chain catabolism in contrast to fatty acid b-oxidation in other species that utilize the evolutionarily distinct crotonase family of hydratases.

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“…The copyright holder for this preprint (which this version posted May 22, 2021. ; https://doi.org/10.1101/2021.05.19.444901 doi: bioRxiv preprint hydration belong to the MaoC family of the thioesterase superfamily, containing a hotdog fold domain, or the Crotonase family (19,(40)(41)(42). These hydratases consist either of a single protein, such as the C 5 side-chain hydratases of P. stutzeri Chol1 and M. tuberculosis (19,42), or of two subunits, such as the C 3 side-chain hydratase of M. tuberculosis (40). Two proteins from the thiolase superfamily with hotdog fold domains are encoded in cluster 2 (Nov2c219 and Nov2c220) adjacent to the ACAD genes.…”

Section: Steroid Transportmentioning

confidence: 99%

Proteome, bioinformatic and functional analyses reveal a distinct and conserved metabolic pathway for bile salt degradation in the Sphingomonadaceae

Feller

Wöhlbrand

Holert

et al. 2021

Preprint

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Bile salts are amphiphilic steroids with a C5 carboxylic side chain with digestive functions in vertebrates. Upon excretion, they are degraded by environmental bacteria. Degradation of the bile-salt steroid skeleton resembles the well-studied pathway for other steroids like testosterone, while specific differences occur during side-chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either Δ1,4- or Δ4,6-3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the Δ1,4- variant is well-known from many model organisms, the Δ4,6-variant involving a 7-hydroxysteroid dehydratase as key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic and functional analyses of the Δ4,6-variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to the Δ1,4-variant except for the elimination of the 7-OH as key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster for side-chain degradation encoding a CoA-ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase, but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the Δ4,6-variant to be widespread among the Sphingomonadaceae, which was verified for three type strains which also have the predicted side-chain degradation cluster. A second amidase in the side-chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile-salt hydrolases. This study signifies members of the Sphingomonadaceae remarkably well-adapted to the utilization of bile salts via a partially distinct metabolic pathway.

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Post-translational Succinylation of Mycobacterium tuberculosis Enoyl-CoA Hydratase EchA19 Slows Catalytic Hydration of Cholesterol Catabolite 3-Oxo-chol-4,22-diene-24-oyl-CoA

Cited by 18 publications

References 53 publications

Enzymatic β-Oxidation of the Cholesterol Side Chain in Mycobacterium tuberculosis Bifurcates Stereospecifically at Hydration of 3-Oxo-cholest-4,22-dien-24-oyl-CoA

Enzymatic β-Oxidation of the Cholesterol Side Chain in Mycobacterium tuberculosis Bifurcates Stereospecifically at Hydration of 3-Oxo-cholest-4,22-dien-24-oyl-CoA

Enzymatic β-Oxidation of the Cholesterol Side Chain in Mycobacterium Tuberculosis Bifurcates Stereospecifically at Hydration of 3-Oxo-Cholest-4,22- Dien-24-Oyl-CoA

Proteome, bioinformatic and functional analyses reveal a distinct and conserved metabolic pathway for bile salt degradation in the Sphingomonadaceae

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