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

Fig 1

Section of the proposed pathway of cholate (compound I) degradation in Pseudomonas sp. strain Chol1. The following compounds have been identified: II, 3-ketocholate; III, Δ^1/4-3-ketocholate (the position of the double bond has not been identified yet; for simplicity, only the more probable Δ⁴ isomer was chosen); IV, Δ^1,4-3-ketocholate; V, cholyl-CoA; VI, 3-ketocholyl-CoA; VII, Δ^1/4-3-ketocholyl-CoA; VIII, Δ^1,4-3-ketocholyl-CoA; IX, CoA ester of (22E)7α,12α-dihydroxy-3-oxochola-1,4-triene-24-oate (DHOCTO); XI, 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA); XII, 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC); XIII, CoA ester of DHOPDC; XVI, 7α,12α-dihydroxy-androsta-1,4-diene-3,17-dione (12α-DHADD); XVII, 7α-hydroxy-androsta-1,4-diene-3,12,17-trione (HADT); XVIII, 12β-DHADD; XIX, 3,7,12-trihydroxy-9,10-secoandrosta-1,3,5(10)triene-9,17-dione (THSATD); XX, DHOCTO; and XXI, 7α,12α22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). The compounds shown in brackets are plausible intermediates that have not been detected yet.

Fig 2

UV spectroscopic and chromatographic properties of the intermediates of cholate degradation. (A) UV spectra of Δ^1,4-3-ketocholate (IV in Fig. 1) (black line), Δ^1,4-3-ketocholyl-CoA (VIII in Fig. 1) (dotted black line), and cholyl-CoA (V in Fig. 1) (gray line) as characteristic examples for steroid compounds with a Δ^1/4- or Δ^1,4-3-keto structure of the A ring (absorption maximum at 245 nm) and for their acyl-CoA esters (absorption maximum at 250 nm), respectively. (B) HPLC chromatogram of a culture supernatant of Pseudomonas sp. strain Chol1 growing with cholate after 8 h of incubation. The analysis wavelength was 245 nm.

Fig 3

HPLC chromatograms of a CoA activation assay with cell extracts of Pseudomonas sp. strain Chol1 containing a mixture of Δ^1/4- and Δ^1,4-3-ketocholate (III and IV in Fig. 1) as the substrates incubated for 0 min (A) and 160 min (B). P1 and P2 represent the CoA esters of Δ^1/4- and Δ^1,4-3-ketocholate (VII and VIII in Fig. 1), respectively, which are hydrolyzed completely after treatment with NaOH (C). The analysis wavelengths were 245 nm (black) and 260 nm (gray).

Fig 4

HPLC chromatograms of CoA activation assays with cell extracts of Pseudomonas sp. strain Chol1 containing DHOCTO (XX in Fig. 1) as the substrate. (A) Representative t₀ for all assays. (B) Assay with nondesalted cell extracts incubated for 60 and 120 min. (C) Assay with desalted cell extracts incubated for 60 min. (D) Assay with desalted cell extracts in the presence of NAD⁺ incubated for 60 and 120 min. P3 was identified as the CoA ester of DHOPDC (XIII), P4 was identified as 12α-DHADD (XVI), P5 was identified as DHOPDCA (XI), and P6 was identified as HADT (XVII). The analysis wavelengths were 245 nm (black) and 260 nm (gray).

Fig 5

HPLC chromatograms of CoA activation assays with cell extracts of Pseudomonas sp. strain Chol1 containing DHOPDC (XII in Fig. 1) as the substrate. (A) Representative t₀ for all assays. (B) Assay with nondesalted cell extracts incubated for 60 and 120 min. (C) Assay with desalted cell extracts incubated for 120 min. (D) Assay with desalted cell extracts in the presence of NAD⁺ incubated for 120 min. (E) Assay with desalted cell extracts in the presence of NAD⁺ and PMS incubated for 120 min. P3 was identified as the CoA ester of DHOPDC (XIII), P4 was identified as 12α-DHADD (XVI), and P6 was identified as HADT (XVII). The analysis wavelengths were 245 nm (black) and 260 nm (gray).

Fig 6

(A) Transformation of DHOPDCA (squares) into DHOPDC (circles) with a desalted cell extract of Pseudomonas sp. strain Chol1 in the presence of NAD⁺ (black lines). Controls without cell extract (dashed gray lines) or without NAD⁺ (solid gray line) showed no transformation of DHOPDCA and no formation of DHOPDC. (B) Chemical and biochemical reactions of DHOPDCA: NAD⁺-dependent oxidation of 20S-DHOPDCA to DHOPDC and chemical transformation of 20S-DHOPDCA to 20R-DHOPDCA under alkaline conditions; the 20R isomer was not oxidized by cell extracts of strain Chol1.

Fig 7

HPLC chromatograms of a CoA activation assay with nondesalted cell extracts of the skt mutant Pseudomonas sp. strain G12 containing DHOCTO (XX in Fig. 1) as the substrate incubated for 0 min (A) and 160 min (B). The analysis wavelengths were 245 nm (gray) and 260 nm (black). P8 was identified as the CoA ester of DHOCTO (IX) and was hydrolyzed completely after treatment with NaOH (C).

Fig 8

Chromatograms of enzyme assays with desalted cell extracts of Pseudomonas sp. strain Chol1 containing P6 as the substrate. (A) Representative t₀ for all assays. (B) Assay with NADH incubated for 30 min. (C) Assay with NADPH incubated for 30 min. P6 was identified as HADT (XVII in Fig. 1). The analysis wavelength was 245 nm. (D) Isomerization of 12α-DHADD to 12β-DHADD via HADT. The oxidation to HADT is NAD⁺ dependent, and the back reaction could be measured in vitro; the reduction of HADT to 12β-DHADD is NADPH dependent; 12β-DHADD is further degraded to THSATD.