Degradation of the Acyl Side Chain of the Steroid Compound Cholate in Pseudomonas sp. Strain Chol1 Proceeds via an Aldehyde Intermediate

Abstract

Bacterial degradation of steroids is widespread, but the metabolic pathways have rarely been explored. Previous studies with Pseudomonas sp. strain Chol1 and the C 24 steroid cholate have shown that cholate degradation proceeds via oxidation of the A ring, followed by cleavage of the C 5 acyl side chain attached to C-17, with 7␣,12␤-dihydroxy-androsta-1,4-diene-3,17-dione (12␤-DHADD) as the product. In this study, the pathway for degradation of the acyl side chain of cholate was investigated in vitro with cell extracts of strain Chol1. For this, intermediates of cholate degradation were produced with mutants of strain Chol1 and submitted to enzymatic assays containing coenzyme A (CoA), ATP, and NAD ؉ as cosubstrates. When the C 24 steroid (22E)-7␣,12␣-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO) was used as the substrate, it was completely transformed to 12␣-DHADD and 7␣-hydroxy-androsta-1,4-diene-3,12,17-trione (HADT) as end products, indicating complete removal of the acyl side chain. The same products were formed with the C 22 steroid 7␣,12␣-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) as the substrate. The 12-keto compound HADT was transformed into 12␤-DHADD in an NADPH-dependent reaction. When NAD ؉ was omitted from assays with DHOCTO, a new product, identified as 7␣,12␣-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA), was formed. This aldehyde was transformed to DHOPDC and DHOPDC-CoA in the presence of NAD ؉ , CoA, and ATP. These results revealed that degradation of the C 5 acyl side chain of cholate does not proceed via classical ␤-oxidation but via a free aldehyde that is oxidized to the corresponding acid. The reaction leading to the aldehyde is presumably catalyzed by an aldolase encoded by the gene skt, which was previously predicted to be a ␤-ketothiolase.

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

“…This pattern is consistent with concurrent side-chain and nucleus degradation by Actinobacteria, as recently established for cholesterol degradation (17,36) and bile acid degradation (27). In contrast, bile acid degradation by Proteobacteria involves complete degradation of the side chain to a keto group, prior to the initial ring cleavage reaction, which opens ring B (9,12). The phylogenetic analysis permits extrapolation of the enzymological data to a broad range of bacteria, since the enzyme classes identified correspond to orthologous groups predicted to have common functions (Fig.…”

“…CasI likely functions in reassimilation of the metabolites which accumulate when RHA1 grows on cholate (27). CasI orthologs likely function in further degradation of an intermediate resulting from the above-mentioned aldolytic cleavage of the cholate side chain by Proteobacteria (12,24). It is unclear whether such aldolytic cleavage occurs in Actinobacteria.…”

“…Steroid side-chain degradation has been studied in both Actinobacteria and Proteobacteria (9,12,13). Nevertheless, the physiological substrates of the enzymes involved in this catabolism are largely unknown.…”

“…Holert et al recently showed evidence for degradation of the cholate side chain in Proteobacteria via a variation of ␤-oxidation, in which an acetyl-CoA moiety is removed by aldolytic, rather than thiolytic, cleavage (12,24). The resulting aldehyde is proposed to be oxidized to an acid and reactivated by CoA thioesterification for another round of ␤-oxidation.…”

“…strain Chol1 (10)(11)(12). Conversely, cholesterol catabolism has been extensively studied in Actinobacteria, especially M. tuberculosis and Rhodococcus species (13)(14)(15)(16)(17).…”