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. Cholesterol is a sterol that modulates membrane fluidity in eukaryotic cells and functions as a hormone precursor. In the liver, cholesterol is converted to bile acids (such as cholate and chenodeoxycholate) by epimerization of the 3␤-hydroxyl group, saturation of the double bond between C-5 and C-6, introduction of hydroxyl groups at the C-7 and C-12 positions, and the shortening of the D-ring side chain from 8 carbon to 5 carbon atoms ( Fig. 1) (1). Bile acids are subsequently excreted in the intestine to aid in the uptake and digestion of lipophilic nutrients. Certain actinobacteria and proteobacteria have the unique ability to grow on cholesterol and cholesterol metabolites, such as bile acids, as sole carbon and energy sources (2-4). Besides its importance in the removal of steroid waste in the environment, the bacterial steroid degradation pathway has received particular attention recently due to the fact that cholesterol is a carbon source for Mycobacterium tuberculosis within host macrophages and disruption of cholesterol degradation genes has been found to attenuate the virulence of the bacteria (5-8).In general, the aliphatic side chain substituent on the D ring of steroids is degraded by reactions analogous to fatty acid ␤-oxidation reactions, involving CoA esterification followed by stepwise removal of 2 or 3 carbon units as acetyl-CoA and propionyl-CoA (9). Due to differences in side chain lengths, cholesterol and cholate side chains will undergo 3 and 2 rounds of ␤-oxidation reactions, respectively. While some bacteria, such as M. tuberculosis, can grow only on cholesterol and not bile acids, the soil actinomycete Rhodococcus jostii RHA1 is able to grow on both cholesterol and cholate (2, 10). R. jostii RHA1 also has separate paralogous genes for degradation of the D-ring aliphatic side chains of these two types of steroids (10).Acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) are key enzymes involved in ␤-oxidation reactions. They are classified based on their substrate specificities (11,12). Those involved in fatty acid ␤-oxidation are the short-, medium-, long-, and verylong-chain acyl-CoA dehydrogenases (SCAD, MCAD, LCAD, and VLCAD, respectively). The other members are involved in