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.

Fig. 1.

Schematic overview of the side chain degradation pathways of cholesterol (A), β-sitosterol and campesterol (B), and steroid ring opening (C) in actinobacteria (6, 10, 36). The arrow numbering indicates reaction steps which are explained in the text. Abbreviations: CoA, coenzyme A; CHO, cholesterol oxidase; 3β-HSD, 3β-hydroxysteroid dehydrogenase; KSTD, 3-ketosteroid Δ1-dehydrogenase; KSH, 3-ketosteroid 9α-hydroxylase.

Fig. 2.

(A) β-Oxidation gene cluster comprised of ro04677 to ro04695 within the cholesterol catabolic gene cluster of R. jostii RHA1 (16, 36). The relative fold change of expression previously observed during growth on cholesterol compared to pyruvate is also shown (36). (B and C) Genetic organization of homologs of ro04688 to ro04690 and ro04691 to ro04695 of R. rhodochrous DSM43269, respectively.

Fig. 3.

HPLC graphs of steroid extracts from β-sitosterol bioconversions after 3 days of incubation. (A) Profile of mutant strain RG32ΔfadD19 showing the accumulation of metabolites (degradation pathway intermediates) that, based on identical HPLC retention times, were identified as 4-sitostene-3-one (I) and 4-campestene-3-one (II). (B) Profile of parent strain RG32.

Fig. 4.

(A) TLC analysis of the reactions of cell extracts (CFE) of E. coli BL21(DE3) cells expressing fadD19_DSM43269, incubated with 5-cholestene-26-oic acid-3β-ol (I), 3-oxo-4-cholestene-26-oic acid (II), and 5-cholenic acid-3β-ol (III). (B) TLC analysis of reactions of CFE of BL21 cells containing empty plasmid pET15b, using the same sterol substrates (I to III). All incubations were performed for 4 h at 30°C and contained the cofactor Mg²⁺. ATP and CoA were either included (+) or omitted (−) as a negative control.