Characterization of Novel Acyl Coenzyme A Dehydrogenases Involved in Bacterial Steroid Degradation

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. 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

Characterization of an Aldolase Involved in Cholesterol Side Chain Degradation in Mycobacterium tuberculosis

“…Protein expression and purification. FadE28-FadE29 and TTHB247 were purified according to the methods described previously (6,14). Recombinant E. coli BL21(DE3) containing chsH1-chsH2, chsH1-chsH2⌬duf35, and duf35 was grown at 37°C in 4 liters of LB medium supplemented with kanamycin (50 g/ml) and/or tetracycline (15 g/ml).…”

“…Precursor ions per cycle were selected for fragmentation. The native molecular masses of purified proteins were estimated using gel filtration on a HiLoad 26/60 Superdex 200 column (GE Healthcare), using a previously described protocol (6).…”

“…heteromeric acyl coenzyme A (acyl-CoA) dehydrogenase that catalyzes dehydrogenation of the C␣-C␤ carbon bond of the 3-carbon-side-chain cholesterol CoA thioester metabolite 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA) (5,6). The genes chsH1 and chsH2, downstream of fadE29, encode a heteromeric enzyme that catalyzes the subsequent hydration reaction (7).…”

Identification of bypass reactions leading to the formation of one central steroid degradation intermediate in metabolism of different bile salts in Pseudomonas sp. strain Chol1

“…Subsequently, a water molecule is added to the double bond, resulting in the formation of a hydroxyl group at C17 of the steroid skeleton, and propionyl-CoA is split off by an aldolytic cleavage reaction, leading to the formation of 12α-DHADD (7α,12α-dihydroxy-androsta-1,4-diene-9,17-dione). strain RHA1 were shown to be ACADs having two fused ACAD domains catalysing the αβ-dehydrogenation reaction of steroid C5 side chain CoA-esters (Ruprecht et al, 2015). In addition, ChsE3 from strain H37Rv and its homologue CasC from the cholate degradation cluster of Rhodococcus jostii sp.…”

“…In addition, ChsE3 from strain H37Rv and its homologue CasC from the cholate degradation cluster of Rhodococcus jostii sp. strain RHA1 were shown to be ACADs having two fused ACAD domains catalysing the αβ-dehydrogenation reaction of steroid C5 side chain CoA-esters (Ruprecht et al, 2015). A heterotetrameric enoyl-CoA hydratase was identified in M. tuberculosis strain H37Rv that catalyses the hydration of an enoyl-CoA C3 side chain intermediate in the course of cholesterol degradation (Yang et al, 2014).…”