Molecular characterization of ltp3 and ltp4, essential for C24-branched chain sterol-side-chain degradation in Rhodococcus rhodochrous DSM 43269

Wilbrink, M.; Geize, Robert van der; Dijkhuizen, Lubbert

doi:10.1099/mic.0.059501-0

Cited by 27 publications

(34 citation statements)

References 40 publications

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“…Interestingly, in Mycobacterium and Rhodococcus, the steroid degradation gene cluster also contained a protein with two tandem DUF35 domains followed by two genes homologous to ltp2 (designated ltp3 and ltp4). Single-gene deletions of ltp3 or ltp4 in R. rhodochrous resulted in mutants that were capable of cholesterol side chain degradation but were deficient in ␤-sitosterol or campesterol side chain degradation (13). Both campesterol and ␤-sitosterol contain branch side chains.…”

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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Section: Discussionmentioning

confidence: 99%

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

2018

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“…5e ) 37 38 . Further, two aldolytic reactions have been proposed and genetically characterized in sterol side-chain degradation, including carbon17-carbon20 cleavage by aldol-lyase Ltp2 39 and C24-branched chain cleavage by aldol-lyases Ltp3 and Ltp4 40 . For many organisms, aldehydes are a group of active compounds that are readily converted to alcohols by reductive enzymatic reactions or to carboxylic acids by oxidative enzymatic reactions.…”

Section: Discussionmentioning

confidence: 99%

Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

Liu

Yao

et al. 2016

Sci Rep

134

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The catabolism of sterols in mycobacteria is highly important due to its close relevance in the pathogenesis of pathogenic strains and the biotechnological applications of nonpathogenic strains for steroid synthesis. However, some key metabolic steps remain unknown. In this study, the hsd4A gene from Mycobacterium neoaurum ATCC 25795 was investigated. The encoded protein, Hsd4A, was characterized as a dual-function enzyme, with both 17β-hydroxysteroid dehydrogenase and β-hydroxyacyl-CoA dehydrogenase activities in vitro. Using a kshAs-null strain of M. neoaurum ATCC 25795 (NwIB-XII) as a model, Hsd4A was further confirmed to exert dual-function in sterol catabolism in vivo. The deletion of hsd4A in NwIB-XII resulted in the production of 23,24-bisnorcholenic steroids (HBCs), indicating that hsd4A plays a key role in sterol side-chain degradation. Therefore, two competing pathways, the AD and HBC pathways, were proposed for the side-chain degradation. The proposed HBC pathway has great value in illustrating the production mechanism of HBCs in sterol catabolism and in developing HBCs producing strains for industrial application via metabolic engineering. Through the combined modification of hsd4A and other genes, three HBCs producing strains were constructed that resulted in promising productivities of 0.127, 0.109 and 0.074 g/l/h, respectively.

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“…The respective substrates of Ltp3, Ltp4, and IpfD contain tertiary ␤-hydroxyl groups, which mechanistically preclude oxidation to the corresponding ␤-keto groups and subsequent thiolytic cleavage. This led to the conclusion that these enzymes catalyze aldolytic cleavage reactions (41). Just like Sal, all three proteins lack the catalytically active N-terminal cysteine, which is highly conserved in catabolic thiolases (43) such as BbsB, which catalyzes a thiolytic cleavage reaction during anaerobic toluene degradation in the denitrifying bacterium Thauera aromatica strain K172 (44), or FadA in E. coli strain K-12 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Genes for three further members of this family, namely, ltp3 and ltp4 from Rhodococcus rhodochrous (41) and ipfD from Sphingomonas sp. strain Ibu-2 (42), were recently revealed to be essential for the acyl side chain degradation of C-24 branched phytosterols and the phenylacetic acid ibuprofen, respectively.…”

Section: Discussionmentioning

confidence: 99%

The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain

2013

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In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C 5 acyl side chain of the C 24 steroid compound cholate involves the C 22 intermediate 7␣,12␣-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C 24 steroid (22E)-7␣,12␣-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7␣,12␣,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A-and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7␣,12␣-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD ؉ as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.

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Molecular characterization of ltp3 and ltp4, essential for C24-branched chain sterol-side-chain degradation in Rhodococcus rhodochrous DSM 43269

Cited by 27 publications

References 40 publications

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

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

Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism

The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain

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