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

The actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode beta-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 ro04690(DSM43269), by gene inactivation studies in mutant strain RG32 of Rhodococcus rhodochrous DSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9 alpha-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 RG32 Delta fadD19, 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 RG32 Delta fadD19 also did not affect cholesterol side chain degradation. Heterologously expressed FadD19(DSM43269) nevertheless was active toward steroid-C-26-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 FadD19(H37Rv) enzymes further suggests that FadD19(H37Rv) has an in vivo role in sterol metabolism of Mycobacterium tuberculosis H37Rv.