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. 2015 Apr;197(8):1360-7.
doi: 10.1128/JB.02420-14. Epub 2015 Feb 2.

Characterization of novel acyl coenzyme A dehydrogenases involved in bacterial steroid degradation

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Characterization of novel acyl coenzyme A dehydrogenases involved in bacterial steroid degradation

Amanda Ruprecht et al. J Bacteriol. 2015 Apr.

Abstract

The acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) FadE34 and CasC, encoded by the cholesterol and cholate gene clusters of Mycobacterium tuberculosis and Rhodococcus jostii RHA1, respectively, were successfully purified. Both enzymes differ from previously characterized ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide. Site-specific mutagenesis showed that only the C-terminal ACAD domain contains the catalytic glutamate base required for enzyme activity, while the N-terminal ACAD domain contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor. Therefore, the two ACAD domains must associate to form a single active site. FadE34 and CasC were not active toward the 3-carbon side chain steroid metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) but were active toward steroid CoA esters containing 5-carbon side chains. CasC has similar specificity constants for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has a preference for the last compound, which has a ring structure similar to that of cholesterol metabolites. Knockout of the casC gene in R. jostii RHA1 resulted in a reduced growth on cholate as a sole carbon source and accumulation of a 5-carbon side chain cholate metabolite. FadE34 and CasC represent unique members of ACADs with primary structures and substrate specificities that are distinct from those of previously characterized ACADs.

Importance: We report here the identification and characterization of acyl-CoA dehydrogenases (ACADs) involved in the metabolism of 5-carbon side chains of cholesterol and cholate. The two homologous enzymes FadE34 and CasC, from M. tuberculosis and Rhodococcus jostii RHA1, respectively, contain two ACAD domains per polypeptide, and we show that these two domains interact to form a single active site. FadE34 and CasC are therefore representatives of a new class of ACADs with unique primary and quaternary structures. The bacterial steroid degradation pathway is important for the removal of steroid waste in the environment and for survival of the pathogen M. tuberculosis within host macrophages. FadE34 is a potential target for development of new antibiotics against tuberculosis.

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Figures

FIG 1
FIG 1
Chemical structures of cholesterol (A) and cholic acid (B) and steroid compounds investigated as potential substrates for ACADs: cholyl-CoA (C), deoxycholyl-CoA (D), 3β-hydroxy-5-cholen-24-oyl-CoA (E), and 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) (F).
FIG 2
FIG 2
Steady-state kinetic analysis of CasC with cholyl-CoA. The initial velocity is shown as a function of cholyl-CoA concentration. The line represents a best fit of the Michaelis-Menten equation to the data.
FIG 3
FIG 3
Growth curves of wild-type and ΔcasC R. jostii RHA1 strains on cholate. Cells of wild-type R. jostii RHA1 (open squares), the wild type containing plasmid pTIPQC1 (triangles), the ΔcasC mutant (circles), and a ΔcasC strain containing the casC gene in plasmid pTIPQC1-His (solid squares) were grown in mineral medium supplemented with 1 mM cholate at 30°C. Growth curves represent the averages of two independent experiments with similar results.
FIG 4
FIG 4
Structure of 2′-pentanoic acid HHIDP.

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