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

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 alpha,12 beta-dihydroxy-androsta-1,4-diene-3,17-dione (12 beta-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 alpha,12 alpha-dihydroxy-3-oxochola-1,4,22-triene-24-oate(DHOCTO) was used as the substrate, it was completely transformed to 12 alpha-DHADD and 7 alpha-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 alpha,12 alpha-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) as the substrate. The 12-keto compound HADT was transformed into 12 beta-DHADD in an NADPH-dependent reaction. When NAD(+) was omitted from assays with DHOCTO, a new product, identified as 7 alpha,12 alpha-dihydroxy-3-oxopregna1,4-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 beta-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 beta-ketothiolase.