Merck
  • Home
  • Search Results
  • Characterization of novel acyl coenzyme A dehydrogenases involved in bacterial steroid degradation.

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

Journal of bacteriology (2015-02-04)
Amanda Ruprecht, Jaymie Maddox, Alexander J Stirling, Nicole Visaggio, Stephen Y K Seah
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. 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Chlorotrimethylsilane solution, 1.0 M in THF
Sigma-Aldrich
Formic acid solution, BioUltra, 1.0 M in H2O
Sigma-Aldrich
Deoxycholic acid, ≥99.0% (T)
Supelco
Acetonitrile, analytical standard
Sigma-Aldrich
Deoxycholic acid, ≥98% (HPLC)
Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Sigma-Aldrich
Acetonitrile, electronic grade, 99.999% trace metals basis
Sigma-Aldrich
Acetonitrile, for DNA synthesis
Sigma-Aldrich
Formic acid, ≥95%, FCC, FG
Supelco
Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material
USP
Residual Solvent Class 2 - Acetonitrile, United States Pharmacopeia (USP) Reference Standard
USP
Levothyroxine, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Formic acid, ACS reagent, ≥96%
Sigma-Aldrich
Formic acid, reagent grade, ≥95%
Sigma-Aldrich
Acetonitrile, ReagentPlus®, 99%
Sigma-Aldrich
Acetonitrile, biotech. grade, ≥99.93%
Sigma-Aldrich
Formic acid, ACS reagent, ≥88%
Sigma-Aldrich
Acetonitrile, suitable for DNA synthesis, ≥99.9% (GC)
Sigma-Aldrich
Acetonitrile, ACS reagent, ≥99.5%
Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Acetonitrile, HPLC Plus, ≥99.9%
Sigma-Aldrich
Ultrapure Acetonitrile
Sigma-Aldrich
Cholic acid, from bovine and/or ovine, ≥98%
Sigma-Aldrich
Nitrilotriacetic acid, Sigma Grade, ≥99%
Sigma-Aldrich
Chlorotrimethylsilane, purified by redistillation, ≥99%
Sigma-Aldrich
Chlorotrimethylsilane, ≥98.0% (GC)
Sigma-Aldrich
Chlorotrimethylsilane, puriss., ≥99.0% (GC)
Sigma-Aldrich
Nitrilotriacetic acid, ACS reagent, ≥99.0%
Sigma-Aldrich
Nitrilotriacetic acid, BioUltra, ≥99.0% (T)