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

Birkenmaier, Antoinette; Möller, Heiko M.; Philipp, Bodo

doi:10.1111/j.1574-6968.2011.02250.x

Cited by 17 publications

(27 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Pseudomonas sp. strain Chol1 and the transposon mutant strains G12 (defective in skt) and R1 (defective in acad) were grown in the phosphate-buffered mineral medium MMChol as described previously (15,18,19). Strain Chol1 was grown with cholate (2 mM), and strains G12 and R1 were grown with mixtures of cholate (2 mM) and succinate (12 mM) in the presence of kanamycin (10 g ml Ϫ1 ).…”

Section: Methodsmentioning

confidence: 99%

“…To induce strain G12 for the transformation of cholate, precultures were grown with 12␤-DHADD (XVIII) (ca. 2 mM) as described previously (18). For the production of 12␤-DHADD, strain Chol1 was grown with cholate under anoxic conditions with nitrate as the electron acceptor, as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

“…In the supernatants of these cultures, either DHOCTO (strain G12) or DHOPDC (strain R1) accumulated along with ⌬ 1/4 -and ⌬ 1,4 -3-ketocholate (III and IV, respectively) in both cases (18,19). Those supernatants were used for the purification of ⌬ 1/4 -and ⌬ 1,4 -3-ketocholate by semipreparative HPLC.…”

mentioning

confidence: 99%

“…The first mutant, strain G12, is defective in the gene skt, which encodes a putative thiolase (18). When this mutant is incubated with cholate, two steroid compounds with a complete C 5 acyl side chain accumulate in culture supernatants, namely, (22E)-7␣,12␣-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO, XX) and 7␣,12␣,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO, XXI).…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

Holert¹,

Kulić²,

Yücel³

et al. 2012

Journal of Bacteriology

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Holert¹,

Kulić²,

Yücel³

et al. 2012

Journal of Bacteriology

View full text Add to dashboard Cite

show abstract

“…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).…”

mentioning

confidence: 99%

Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp

et al. 2012

View full text Add to dashboard Cite

bBile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as well as on its conjugates, taurocholate and glycocholate. The transcriptome of RHA1 growing on cholate revealed 39 genes upregulated on cholate, occurring in a single gene cluster. Reverse transcriptase quantitative PCR confirmed that selected genes in the cluster were upregulated 10-fold on cholate versus on cholesterol. One of these genes, kshA3, encoding a putative 3-ketosteroid-9␣-hydroxylase, was deleted and found essential for growth on cholate. Two coenzyme A (CoA) synthetases encoded in the cluster, CasG and CasI, were heterologously expressed. CasG was shown to transform cholate to cholyl-CoA, thus initiating side chain degradation. CasI was shown to form CoA derivatives of steroids with isopropanoyl side chains, likely occurring as degradation intermediates. Orthologous gene clusters were identified in all available Rhodococcus genomes, as well as that of Thermomonospora curvata. Moreover, Rhodococcus equi 103S, Rhodococcus ruber Chol-4 and Rhodococcus erythropolis SQ1 each grew on cholate. In contrast, several mycolic acid bacteria lacking the gene cluster were unable to grow on cholate. Our results demonstrate that the above-mentioned gene cluster encodes cholate catabolism and is distinct from a more widely occurring gene cluster encoding cholesterol catabolism. Bile salts are surface-active steroids with an important role in the uptake and metabolism of lipophilic substrates in vertebrates. These steroids, which include cholate and chenodeoxycholate, are synthesized in the liver from cholesterol, and their eventual fate is excretion in feces or urine. Bile salts may be modified, either by microbiological activity in the duodenum or by host cell bioactivity, leading to their conjugation to glycine, taurine, or sulfate. As such, biodegradation of the various bile salts is a significant process in carbon cycling in soil and aquatic environments. The processes involved in microbial transformation of steroids are also relevant for biotechnological applications in the synthesis and/or selective modification of steroid-based drugs (29).Despite the cytotoxicity of cholate toward various prokaryotic and eukaryotic cells, several bacterial species, especially members of the Proteobacteria (27, 28) and Actinomycetales (9, 32), are able to efficiently metabolize this substrate to sustain growth. Recent studies on microbial bile salts degradation have focused on the Proteobacteria. Genes encoding several steps in cholate degradation were identified, mainly in Comamonas testosteroni TA441 and Pseudomonas sp. strain Chol1. In the former strain, genes responsible for oxidation of the steroid nucleus were found (10-13), while in the latter, genes responsible for degradation of the cholate side chain were identified, in...

show abstract