Microbial degradation of the phytosterol side chain. II. Incorporation of [14C]-NaHCO3 onto the C-28 position

Fujimoto, Yoshinori; Chen, Ching Shih; Gopalan, Aravamudan S.; Sih, Charles J.

doi:10.1021/ja00381a056

Cited by 32 publications

(13 citation statements)

References 3 publications

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“…This process has been elucidated at the biochemical level in strains of Mycobacterium and Nocardia and proceeds differently for ␤-sitosterol than for cholesterol ( Fig. 1) (9,10,13,14,(27)(28)(29).…”

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confidence: 99%

FadD19 of Rhodococcus rhodochrous DSM43269, a Steroid-Coenzyme A Ligase Essential for Degradation of C-24 Branched Sterol Side Chains

Wilbrink

Petrusma

Dijkhuizen

et al. 2011

Appl Environ Microbiol

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The actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode β-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e., fadD17, fadD19, fadE26, fadE27, and ro04690DSM43269, by gene inactivation studies in mutant strain RG32 of Rhodococcus rhodochrous DSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of five kshA gene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion of fadD19 in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation of fadD17 in mutant RG32ΔfadD19 also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269 nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essential in vivo role in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with an in vivo role in sterol side chain degradation. The high similarity (67%) between the FadD19(DSM43269) and FadD19H37Rv enzymes further suggests that FadD19H37Rv has an in vivo role in sterol metabolism of Mycobacterium tuberculosis H37Rv.

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FadD19 of Rhodococcus rhodochrous DSM43269, a Steroid-Coenzyme A Ligase Essential for Degradation of C-24 Branched Sterol Side Chains

Wilbrink

Petrusma

Dijkhuizen

et al. 2011

Appl Environ Microbiol

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“…1. Overview of the pathways involved in sterol degradation in actinobacteria (adapted from Sih et al, 1968b;Fujimoto et al, 1982b;van der Geize et al, 2007). (a) Cholesterol-side-chain degradation is initiated by formation of a cholesterol C26-oic acid (1), involving CYP125 (Rosłoniec et al, 2009).…”

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confidence: 99%

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

2012

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A previously identified sterol catabolic gene cluster is widely dispersed among actinobacteria, enabling them to degrade and grow on naturally occurring sterols. We investigated the physiological roles of various genes by targeted inactivation in mutant RG32 of Rhodococcus rhodochrous, which selectively degrades sterol side-chains. The ltp3 and ltp4 deletion mutants were each completely blocked in side-chain degradation of b-sitosterol and campesterol, but not of cholesterol. These results indicated a role for ltp3 and ltp4 in the removal of C24 branches specifically. Bioinformatic analysis of the encoded Ltp3 and Ltp4 proteins revealed relatively high similarity to thiolase enzymes, typically involved in b-oxidation, but the catalytic residues characteristic for thiolase enzymes are not conserved in their amino acid sequences. Removal of the C24-branched side-chain carbons of b-sitosterol was previously shown to proceed via aldolytic cleavage rather than by b-oxidation. Our results therefore suggest that ltp3 and ltp4 probably encode aldol-lyases rather than thiolases. This is the first report, to our knowledge, on the molecular characterization of genes with specific and essential roles in carbon-carbon bond cleavage of C24-branched chain sterols in Rhodococcus strains, most likely acting as aldollyases. The results are a clear contribution to our understanding of sterol degradation in actinobacteria.

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“…As distinct from cholesterol, cleavage of a PS with a branched lateral chain at carbon 24 involves HC [11][12][13]. Cleavage of bonds between carbons 24 and 25, as well as between carbons 24 and 28 in the lateral chain of PS, is preceded by the introduction of the hydrocarbonate ion at carbon 28 ( Fig.…”

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“…Its enzymatic introduction into the steroid molecule was confirmed by incubation of 3-keto-24-ethylcholest-4-en-26-ol acid with a cell-free extract from AD-producing Mycobacterium sp. B-3805 in the presence of labeled CO 2 , ATP, and coenzyme A [12,13]. This work was designed to optimize the conditions for PS fermentation, which provided for saturation of the culture liquid in the AD-producing strain of Mycobacterium neoaurum with carbon dioxide.…”

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confidence: 99%

10.1007/s10438-008-1008-0

2000

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Microbial degradation of the phytosterol side chain. II. Incorporation of [14C]-NaHCO3 onto the C-28 position

Cited by 32 publications

References 3 publications

FadD19 of Rhodococcus rhodochrous DSM43269, a Steroid-Coenzyme A Ligase Essential for Degradation of C-24 Branched Sterol Side Chains

FadD19 of Rhodococcus rhodochrous DSM43269, a Steroid-Coenzyme A Ligase Essential for Degradation of C-24 Branched Sterol Side Chains

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

10.1007/s10438-008-1008-0

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