title: FadD3 is an acyl-CoA synthetase that initiates catabolism of cholesterol rings C and D in actinobacteria.
abstract: The cholesterol catabolic pathway occurs in most mycolic acid-containing actinobacteria, such as Rhodococcus jostii RHA1, and is critical for Mycobacterium tuberculosis (Mtb) during infection. FadD3 is one of four predicted acyl-CoA synthetases potentially involved in cholesterol catabolism. A ΔfadD3 mutant of RHA1 grew on cholesterol to half the yield of wild-type and accumulated 3aα-H-4α(3'-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), consistent with the catabolism of half the steroid molecule. This phenotype was rescued by fadD3 of Mtb. Moreover, RHA1 but not ΔfadD3 grew on HIP. Purified FadD3(Mtb) catalysed the ATP-dependent CoA thioesterification of HIP and its hydroxylated analogues, 5α-OH HIP and 1β-OH HIP. The apparent specificity constant (k(cat) /K(m) ) of FadD3(Mtb) for HIP was 7.3 ± 0.3 × 10(5)  M(-1)  s(-1) , 165 times higher than for 5α-OH HIP, while the apparent K(m) for CoASH was 110 ± 10 μM. In contrast to enzymes involved in the catabolism of rings A and B, FadD3(Mtb) did not detectably transform a metabolite with a partially degraded C17 side-chain. Overall, these results indicate that FadD3 is a HIP-CoA synthetase that initiates catabolism of steroid rings C and D after side-chain degradation is complete. These findings are consistent with the actinobacterial kstR2 regulon encoding ring C/D degradation enzymes.
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver V6T 1Z3, Canada.
authors.forename: Israël
authors.initials: I
authors.lastname: Casabon
authors.forename: Adam M
authors.initials: AM
authors.lastname: Crowe
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
mesh_terms: D002784:Cholesterol / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D002784:Cholesterol; D003066:Coenzyme A Ligases