Identification of a thiolase gene essential for p-oxidation of the acyl side chain of the steroid compound cholate in Pseudomonassp. strain Chol1

Bile salts such as cholate are steroid compounds occurring ubiquitously in the environment through excretion by animals. Cholate degradation by Pseudomonas sp. strain Choll is initiated by A ring oxidation and ~ oxidation of the acyl side chain. A transposon mutant of strain Choll was isolated that could not grow with cholate, but transformed it into several steroid compounds accumulating in culture supernatants. The main product was identified as (22E) 7cx,12cx dihy droxy 3 oxochola 1,4,22 triene 24 oate (DHOCTO). A further compound was identified as 7cx,12cx,22 trihydroxy 3 oxochola 1,4 diene 24 oate (THOCDO). The structures of DHOCTO and THOCDO indicate that they are intermediates of the ~ oxidation of the acyl side chain. The interrupted gene was named skt and had similarities to the 3 keto acyl CoA thiolase domain of the eukaryotic sterol carrier protein SCP x. An skt mutant grew with intermediates of cholate degrada tion, from which the acyl side chain had been partly or completely removed. Growth with cholate was restored by an intact skt copy on a plasmid. These results strongly suggest that skt encodes a ~ ketothiolase responsible for the cleavage of acetyl CoA from the acyl side chain of cholate. Sequence comparisons revealed that other steroid degrading bacteria such as Coma monas testosteroni contain genes encoding proteins very similar to Skt, suggesting a widespread role of this enzyme in bacterial steroid degradation.