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FadD 3 is an acyl‐ CoA synthetase that initiates catabolism of cholesterol rings C and D in actinobacteria
Author(s) -
Casabon Israël,
Crowe Adam M.,
Liu Jie,
Eltis Lindsay D.
Publication year - 2013
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12095
Subject(s) - catabolism , biochemistry , biology , fadd , enzyme , steroid , mutant , metabolite , actinobacteria , gene , apoptosis , 16s ribosomal rna , programmed cell death , hormone , caspase
Summary The cholesterol catabolic pathway occurs in most mycolic acid‐containing actinobacteria, such as R hodococcus jostii RHA 1, and is critical for M ycobacterium tuberculosis ( Mtb ) during infection. FadD 3 is one of four predicted acyl‐ CoA synthetases potentially involved in cholesterol catabolism. A Δ fadD3 mutant of RHA 1 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, RHA 1 but not Δ fadD3 grew on HIP . Purified FadD 3 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 FadD 3 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 μ M . In contrast to enzymes involved in the catabolism of rings A and B , FadD 3 Mtb did not detectably transform a metabolite with a partially degraded C 17 side‐chain. Overall, these results indicate that FadD 3 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.