Identification of Genes Involved in Inversion of Stereochemistry of a C-12 Hydroxyl Group in the Catabolism of Cholic Acid by Comamonas testosteroni TA441

Abstract

Comamonas testosteroni TA441 degrades steroids such as testosterone via aromatization of the A ring, followed by meta-cleavage of the ring. In the DNA region upstream of the meta-cleavage enzyme gene tesB, two genes required during cholic acid degradation for the inversion of an ␣-oriented hydroxyl group on C-12 were identified. A dehydrogenase, SteA, converts 7␣,12␣-dihydroxyandrosta-1,4-diene-3,17-dione to 7␣-hydroxyandrosta-1,4-diene-3,12,17-trione, and a hydrogenase, SteB, converts the latter to 7␣,12␤-dihydroxyandrosta-1,4-diene-3,17-dione. Both enzymes are members of the short-chain dehydrogenase/reductase superfamily. The transformation of 7␣,12␣-dihydroxyandrosta-1,4-diene-3,17-dione to 7␣,12␤-dihydroxyandrosta-1,4-diene-3,17-dione is carried out far more effectively when both SteA and SteB are involved together. These two enzymes are encoded by two adjacent genes and are presumed to be expressed together. Inversion of the hydroxyl group at C-12 is indispensable for the subsequent effective B-ring cleavage of the androstane compound. In addition to the compounds already mentioned, 12␣-hydroxyandrosta-1,4,6-triene-3,17-dione and 12␤-hydroxyandrosta-1,4,6-triene-3,17-dione were identified as minor intermediate compounds in cholic acid degradation by C. testosteroni TA441. Rhodococcus equi and Comamonas testosteroni (formerlyNocardia restrictus and Pseudomonas testosteroni) are known for the ability to utilize testosterone and various other steroids, such as the cholic acid analogs. In the 1950s and 1960s, the mechanism by which testosterone is degraded in these bacteria was extensively studied, and the main intermediate compounds in the degradation pathway were determined (2-6, 16-19). In our previous work, we simultaneously identified the genes and the intermediate compounds that are accumulated by gene disruption mutants and revealed the testosterone degradation pathway and degradation genes of C. testosteroni TA441 (7-13). Two steroid degradation gene clusters were identified in TA441; one contains the meta-cleavage enzyme gene tesB, 16 ORFs, and the positive regulator of the steroid degradation gene tesR, and the other consists of ORF18, ORF17, and tesIHA2A1DEFG. TA441 degrades testosterone via aromatization of the A ring, followed by cleavage of the ring by enzymes encoded by these genes. Most of the enzymes involved in the transformation of testosterone to 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid are encoded by the ORF18-tesG gene cluster. ORF18 has been reported to encode a CoA transferase which adds CoA to 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, and most of the putative ORFs in the region downstream of tesB have been suggested to be involved in 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid degradation, which is thought to be similar to the ␤-oxidation pathway of fatty acids. However, the region upstream of tesB was not clear. In this report, we describe newly isolated genes essential for the degradation of 12␣-hydroxylated ...

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

“…This could be the reason for the fact that strain R1 is not able to grow with cholate as the sole carbon source, despite its ability to release one acetyl-CoA residue from the substrate (19). The propionyl residue was further degraded via the methylcitric acid cycle, which is supported by the fact that all genes for this pathway are present in the genome of strain Chol1, while those for the methylmalonyl-CoA pathway are not (32).…”

“…The function of this specificity for the reduction of HADT is not known. In C. testosteroni, the formation of 12␤-DHADD is necessary for the subsequent hydroxylation at C-9 and the formation of THSATD (32). The same might be true for strain Chol1, because 12␤-DHADD, the end product of anaerobic cholate degradation, is immediately transformed into THSATD by cell suspensions upon aeration (15).…”

“…The isomerization of 12␣-DHADD (XVI) to 12␤-DHADD (XVIII) via HADT (XVII) has also been observed during cholate degradation by Comamonas testosteroni strain TA441 and is catalyzed by the consecutive action of a dehydrogenase (SteA) and a reductase (SteB) (32). Genes (ORF_11442 and ORF_11447) encoding predicted proteins with high similarities to SteA (58%) and SteB (70%), respectively, are also found in the genome of strain Chol1 (30).…”

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp

“…strain Chol1. In the former strain, genes responsible for oxidation of the steroid nucleus were found (10)(11)(12)(13), while in the latter, genes responsible for degradation of the cholate side chain were identified, including acad, encoding an acyl coenzyme A (acyl-CoA) dehydrogenase (2), and skt, encoding a thiolase (3).…”

Identification of 9 -Hydroxy-17-Oxo-1,2,3,4,10,19-Hexanorandrostan-5-Oic Acid in Steroid Degradation by Comamonas testosteroni TA441 and Its Conversion to the Corresponding 6-En-5-Oyl Coenzyme A (CoA) Involving Open Reading Frame 28 (ORF28)- and ORF30-Encoded Acyl-CoA Dehydrogenases

“…The main intermediate compounds in bacterial steroid degradation were identified from the 1950s through the 1980s, and the mechanism of steroid degradation was predicted on the basis of the compounds identified for the purpose of obtaining medical materials (1)(2)(3)(4)(5)(6)(7)(8)(9). The degradation genes have been known since approximately 1980 (10)(11)(12)(13)(14)(15)(16)(17)(18)(19), but the details of the degradation had remained unclarified, until we revealed the degradation mechanism of the A and B rings of the core steroidal structure (20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Our studies have suggested that an approximately 100-kb DNA region in C. testosteroni TA441 is a large steroid degradation gene cluster (Fig.…”