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

Abstract

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.

Deciphering the Transcriptional Regulation of Cholesterol Catabolic Pathway in Mycobacteria

“…After complete oxidation of C26 to the corresponding carboxylic acid, the cholesterol side chain is shortened by a process similar to ␤-oxidation of fatty acids (22,50,51). This process is initiated by a steroid CoA ligase, which catalyzes the CoA activation of the C26 carboxylic acid intermediates (51,52).…”

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

“…FadD19 occurs in all actinobacterial cholesterol degradation pathways characterized to date (25,26,29,45). So far, Proteobacteria have not been reported to catabolize cholesterol, and accordingly, they lack orthologs of FadD19.…”

“…One exception to this pattern is Comamonas testosteroni CNB-2, which has a gene of unknown function encoding a protein that shares 37% amino acid sequence identity with FadD19 RHA1 . FadD19 of R. rhodochrous DSM 43269 was also shown to transform cholestenoates, but it is not essential for cholesterol degradation (29). On the other hand, FadD19 of M. tuberculosis is essential for cholesterol degradation (31).…”

“…S1 in the supplemental material) (26). Although there is no report about FadD17 or FadD19 of RHA1, their orthologs from M. tuberculosis have been shown to catalyze the CoA thioesterification of long-chain fatty acids (LCFAs) (28), while FadD19 from Rhodococcus rhodochrous DSM 43269 is involved in degradation of C 24 branched-chain sterols (29). Additionally, FadD3 was recently identified as being essential for the catabolism of cholesterol and cholate rings C and D in RHA1 (14), and disruption of orf18, encoding a homolog of FadD3 in C. testosteroni TA441, abrogates degradation of bile acid rings C and D (30).…”

Deletion of the gene encoding the reductase component of 3-ketosteroid 9¿-hydroxylase in

“…Bacterial strains belonging to Mycobacterium, Rhodococcus, Nocardia and Arthrobacter genera are known for their ability to degrade a range of naturally occurring steroids. The catabolic pathways for sterols have been proposed based on the results of a large body of biochemical and genomic studies [2][3][4]. A simplified illustration emphasizing biotransformation of AD and ADD is shown in Figure 1.…”