Biosynthesis of a novel bile acid nucleotide and mechanism of 7 alpha-dehydroxylation by an intestinal Eubacterium species.

Eubacterium species V.P.I. 12708 has inducible bile acid 7-dehydroxylase activity that can use either 7 alpha or 7 beta bile acids as substrates. Cell extracts prepared from bacteria grown in the presence of cholic acid catalyzed the rapid conversion of free bile acids into a highly polar bile acid metabolite (HPBA). This conjugation activity co-eluted with bile acid 7-dehydroxylase activity on high performance gel filtration chromatography (GFC). The HPBA was purified by a combination of high performance GFC and reverse-phase high performance liquid chromatography (HPLC). The intact HPBA eluted earlier from reverse-phase HPLC than deoxycholyl-CoA and had a Mr of 1102 by Bio-Gel P-2 (GFC). The HPBA had an absorption peak at 255 nm and was sensitive to treatment with phosphodiesterase I or nucleotide pyrophosphatase. The HPBA has a free phosphate as shown by an increase in elution volume on reverse-phase HPLC following treatment with alkaline phosphatase. Treatment of the purified HPBA with nucleotide pyrophosphate plus alkaline phosphatase yielded adenosine, whereas, treatment with nucleotide pyrophosphatase alone generated 5',3'-ADP. A bile acid metabolite was also generated by nucleotide pyrophosphatase treatment. The bile acid metabolite had different chromatographic properties (HPLC and TLC) than the corresponding free bile acid. Gas liquid chromatography-mass spectrometry showed the bile acid metabolite to be 12 alpha-hydroxy-3-oxo-4-cholenoic acid. We hypothesize that the HPBA is an intermediate in 7-dehydroxylation and consists of this compound linked at the C-24 with an anhydride bond to the beta phosphate (5') of ADP-3'-phosphate. These results suggest a novel mechanism of bile acid 7 alpha/7 beta-dehydroxylation in Eubacterium sp. V.P.I. 12708.

Eubacterium species V.P.I. 12708 has inducible bile acid 7-dehydroxylase activity that can use either 7a or 78 bile acids as substrates. Cell extracts prepared from bacteria grown in the presence of cholic acid catalyzed the rapid conversion of free bile acids into a highly polar bile acid metabolite (HPBA). This conjugation activity co-eluted with bile acid 7-dehydroxylase activity on high performance gel filtration chromatography (GFC). The HPBA was purified by a combination of high performance GFC and reverse-phase high performance liquid chromatography (HPLC). The intact HPBA eluted earlier from reverse-phase HPLC than deoxycholyl-CoA and had a M, of 1102 by Bio-Gel P-2 (GFC). The HPBA had an absorption peak at 255 nm and was sensitive to treatment with phosphodiesterase I or nucleotide pyrophosphatase. The HPBA has a free phosphate as shown by an increase in elution volume on reverse-phase HPLC following treatment with alkaline phosphatase. Treatment of the purified HPBA with nucleotide pyrophosphate plus alkaline phosphatase yielded adenosine, whereas, treatment with nucleotide pyrophosphatase alone generated 5',3'-ADP. A bile acid metabolite was also generated by nucleotide pyrophosphatase treatment. The bile acid metabolite had different chromatographic properties (HPLC and TLC) than the corresponding free bile acid. Gas liquid chromatography-mass spectrometry showed the bile acid metabolite to be 12a-hydroxy-3-oxo-4-cholenoic acid. We hypothesize that the HPBA is an intermediate in 7-dehydroxylation and consists of this compound linked at the C-24 with an anhydride bond to the 8 phosphate ( 5 ' ) of ADP-3'-phosphate. These results suggest a novel mechanism of bile acid 7a/7@-dehydroxylation in Eubacterium sp. V.P.I. 12708.

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In man, the intestinal microflora can generate at least 15 to 20 different bile acids from the primary bile acids, cholic acid and chenodeoxycholic acid (1-4). Quantitatively, the most important bile acid biotransformation is the 7a-dehydroxylation of cholic acid and chenodeoxycholic acid yielding deoxycholic acid and lithocholic acid, respectively. Deoxycholic acid and to a lesser extent lithocholic acid are absorbed from the colon and returned to the liver where they are 11 To whom correspondence should be addressed. conjugated to either taurine or glycine. These two secondary bile acids constitute 20 to 25% of the total biliary bile acid We have previously reported that Eubacterium sp. V.P.I. 12708 has an inducible bile acid 7a-dehydroxylase activity (5). Enzyme activity was highly stimulated by the addition of NAD+ to cell extracts as was the reduction of 3a-hydroxy-50chol-6-enoic acid (A6), an intermediate in this biotransformation (6-8). Bile acid substrate specificity studies showed that 7-dehydroxylase from this bacterium was highly specific, requiring a free C-24 carboxyl group and an unhindered 7aor 7/3-hydroxy group for activity. The relative molecular weight of bile acid 7-dehydroxylase was 114,000 as estimated by gel filtration chromatography. Both 7a-and 70-dehydroxylase and A6-reductase activities co-eluted suggesting that all three activities may reside in the same protein or enzyme complex. However, efforts to further purify 7adehydroxylase by activity measurements were unsuccessful due to the apparent lability of this enzyme.
In the current article, we report the discovery and identification of a novel bile acid nucleotide formed by cell extracts of Eubacterium sp. V.P.I. 12708 when grown in the presence of primary bile acids. pool.

RESULTS
Formation of Highly Water Soluble Bile Acids-We discovered that in enzymatic assays for bile acid 7-dehydroxylase activity a small but constant percent of added [24-'4C]cholic acid or [24-'4C]deoxycholic acid became resistant to extraction by ethyl acetate. The amount of [24-14C]bile acid found in the aqueous phase after ethyl acetate extraction was at least 13-fold greater in cell extracts prepared from cholic acidinduced cultures as compared to control cultures (no added bile acids). The amount of highly water soluble bile acids (HSBA)' was linearly (0.25 to 1 mg) dependent upon the concentration of extract protein (Fig. 1, see Miniprint). The initial reaction rate was very rapid (Fig. 2, see Miniprint) and the amount of HSBA remained constant after about 60 s. The formation of HSBA by cell extracts was sensitive to heating Portions of this paper (including "Experimental Procedures," Figs. 1, 2, 5, and 7, and Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-2860, cite the authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
* The abbreviations used are: HSBA, highly water soluble bile acide, HPLC, high performance liquid chromatography; RI, retention index.

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(95 "C for 5 min) and prior treatment with trypsin (data not shown).
In order to determine if a specific protein(s) was catalyzing this conjugation reaction, cell extract from cholic acid-induced cultures was fractionated by high performance gel filtration chromatography and individual fractions assayed for the formation of HSBA. The results (Fig. 3) (Table I).
HPLC of Highly Water Soluble Bib Acid Conjugate-Initially HSBA was chromatographed on a Cl8 reverse-phase HPLC column using a MeOH-HZ0 gradient solvent system. The data in Fig. 4 shows the elution profile of [24-'4C]cholic HSBA (XCA) relative to cholic acid, glycocholic acid, and taurocholic acid standards. The HSBA eluted very rapidly from the Cl8 reverse-phase column with a polarity greater than that of taurocholic acid. Efforts to purify HSBA from reaction mixtures were next undertaken.
"C]deoxycholate was first injected onto a DuPont GF-250 high performance gel filtration column, fractions (1.0 ml) were collected and counted for radioactivity.
Peaks 1 (Fraction 14) and 2 (Fraction 20) contained HSBA and peak 3 (Fraction 29) contained ethyl acetate-extractable bile acids. However, thin layer chromatography of the bile acids in peak 3 showed .them to migrate faster than standard [24-14C]deoxycholic acid. HSBA contained in peaks 1 and 2 were further purified by Cl, reverse-phase HPLC using an isopropyl alcohol gradient (5% to 40%) solvent system in ammonium/carbonate buffer (pH 7.0). The data in Fig. 6 shows the elution profile of peak 1 monitored at 254 nm from bile acid-induced cultures. Peaks at 28.3 and 29.3 min were consistently observed from induced cell extracts and contained all bile acid radioactivity. The elution time of authentic standard deoxycholyl-CoA was approximately 33 min. The two UV absorbing peaks were pooled and scanned for their absorption maximum. The data in Fig. 7 (see Miniprint) showed an absorption peak at 255 nm. Enzymatic Treatment of HSBA-The HPLC-purified HSBA (Fig. 6) was next treated with different enzymes which recognize phosphate or nucleotides and the products chromatographed using reverse-phase HPLC using the isopropyl alcohol (5% to 40%) gradient system described in the legend to Fig. 6. Products were monitored for radioactivity and absorption at 254 nm. The only enzymes that were found to degrade HSBA were alkaline phosphatase, phosphodiesterase 1, and nucleotide pyrophosphatase. Treatment of purified HSBA with alkaline phosphatase altered the elution volume from approximately 30 to 34 ml (Fig. 8,panel B). Radioactivity and the 254-nm absorbing material co-eluted in the same fractions. In contrast, treatment of the HSBA with phosphodiesterase 1 resulted in a marked decrease in absorption at 254 nm and a shift in the elution of radioactivity from 30 to 36.5 ml (Fig. 8, panel C). However, it should be noted that the bile acid metabolite retained some absorbance at 254 nm. Treatment of purified HSBA with nucleotide pyrophosphatase also resulted in a marked decrease in absorption at 254 nm and an increase in the elution time from 30 to 36.5 ml (data not shown). Under identical chromatographic conditions [24-14C]deoxycholic acid eluted at 42 ml.
Identification of the Nucleotide Linked to Deoxycholic Acid-The identification of the nucleoside linked to bile acid was determined by treating the purified bile acid nucleotide with a combination of nucleotide pyrophosphatase plus alkaline phosphatase. The nucleoside released was identified by reverse-phase HPLC as adenosine (Fig. 9). The phosphate orientations of the bile acid nucleotide were established by treating it with nucleotide pyrophosphatase. The nucleotide released eluted (6.94 min) at essentially the same time (6.92 min) as the 3',5'-ADP standard (Fig. 10, panel B). Small amounts of 5'-AMP were also generated, possibly due to chemical breakdown of the nucleotide or trace contamination by phosphatases in nucleotide pyrophosphatase. The orientation of the pyrophosphate linkage was confirmed by initially treating the purified bile acid nucleotide with alkaline phosphatase followed by purification using the solvent system described in the legend to Fig. 6 and then treatment with nucleotide pyrophosphatase. The results in Fig. 10, panel C, showed the formation of AMP (4.87 min) after nucleotide pyrophosphatase treatment, showing that the pyrophosphate linkage is at the 5' position.

P-2 Gel Filtration Chromatography of Bile Acid Nucleotide-
The relative molecular weight of the intact bile acid nucleotide and the radiolabeled product generated by phosphodiesterase and alkaline phosphatase treatment were estimated by Bio-Gel P-2 gel filtration chromatography. The bile acid nucleotide and the phosphodiesterase generated radiolabeled product had relative molecular weights of 1102 and 508, respectively (Fig. 11). Alkaline phosphatase treatment generated a product with a relative molecular weight of approximately 904.
Identification of Bile Acid Moiety of HSBA-Phosphodiesterase 1 and nucleotide pyrophosphatase treatment of the purified HSBA generated two products: a nucleotide and a radiolabeled bile acid metabolite. The nucleotide was identified as 5I-pA-3"~. The bile acid metabolite migrated with a higher polarity than deoxycholic acid by reverse-phase HPLC (Fig. 8, panel C). In contrast, the bile acid migrated faster than authentic deoxycholic acid on TLC (Fig. 12). A deoxycholic acid metabolite with the same R F on TLC as generated The intact deoxycholyl nucleotide or the purified alkaline phosphatase-treated material (Fig. 8, paneki A and B ) was treated with nucleotide pyrophosphatase (0.1 unit, 37 "C for 15 min) and the nucleotide released separated and identified using CIS reverse-phase ion pair HPLC. A solvent system of 19% acetonitrile in 0.03 M KH~POI with 0.01 M tetrabutylammonium phosphate (pH 2.65). The flow rate was 1 ml/min. The injection volume was 1.0 ml and the UV detector was operated at 0.1 absorbance units at full scale (254 nm). The data in panel A show the relative elution rates of AMP (4.89 min), 5',2'-ADP (6.43 min), 5',3'-ADP (6.92 min), and ATP (13.14 min) standards. well as the alkaline phosphatase (ZI) and phosphodiesterase 1 (1II)treated material was purified by HPLC (Fig. 6). Approximately 20,000 dpm of each was applied to the P-2 gel filtration column, fractions were (2.5 ml) collected, and radioactivity determined by liquid scintillation counting. The column was calibrated using vitamin B-12 by phosphodiesterase 1 treatment was observed to accumulate in ethyl acetate extracts of conjugate reaction mixtures. The deoxycholic acid metabolite from phosphodiesterase 1 treatment was added back to soluble cell extracts but was not converted into HSBA.
When subjected to anion exchange chromatography on Lipidex DEAP, the deoxycholic acid metabolite was eluted A. t' .,: -" B C FIG. 12. Thin layer chromatography of deoxycholic acid metabolite (DX). Purified bile acid nucleotide was treated with phosphodiesterase 1 (1 unit) and deoxycholic acid metabolite purified by HPLC (Fig. 8, panel C). The radiolabeled deoxycholate acid metabolite was spotted on TLC and separated as described under "Experimental Procedures." Cholate (C), deoxycholate (D), and lithocholate (L) standards were chromatographed under identical conditions. The origin (0) and solvent front (F) are indicated. with 0.1 M acetic acid in 70% aqueous ethanol, thus having the mobility of a conventional unconjugated bile acid (12). The negative ion fast atom bombardment-mass spectrum obtained before and after ion exchange chromatography showed a prominent ion at m/z 387, interpreted to be the quasimolecular ion ["I]-of a deoxycholic acid metabolite that had lost four hydrogen atoms.
Methylation with diazomethane and treatment with trimethylchlorosilane/hexamethyldisilazane/pyridine, 1:2:3 (by volume), yielded a compound whose retention index (RI) on the methyl silicone capillary column was 3324. The mass spectrum showed a molecular ion at m/z 474 indicating the derivative of an unsaturated monohydroxy-monooxocholanoate. Peaks characteristic of a 12-oxo group were absent indicating that the oxo group was at C-3 (13).
Treatment of the methyl ester with hydroxylammonium chloride in pyridine followed by trimethylsilylation yielded two compounds with RI 3363 and 3379, respectively, in proportions 1:2. This indicated formation of syn (RI 3363) and anti (RI 3379) isomers of a 3-oxo-A4-steroid structure (14). The mass spectra, showing a prominent ion at m/z 211 (13), supported this interpretation. The derivatives of 12a-hydroxy-3-oxo-4-cholenoic acid were therefore analyzed in the same way and retention indices and mass spectra were identical to those of the derivatives of the bile acid moiety of HSBA.

DISCUSSION
Data presented in this study shows that the HSBA formed by whole cells or cell extracts of Eubacterium sp. V.P.I. 12708 is a novel bile acid adenosine nucleotide. The purified HSBA had an absorption peak at approximately 255 nm in buffer and was sensitive to degradation by phosphodiesterase 1 or nucleotide pyrophosphatase. These results strongly suggest the presence of a nucleotide with a pyrophosphate group covalently linked to the bile acid. Substrate specificity and competition experiments (Table I) strongly suggest that the nucleotide is covalently linked to the C-24 carboxyl group.
Treatment of the HSBA with nucleotide pyrophosphatase generated a nucleotide (5'-pA-3'-p) and a deoxycholic acid metabolite. Gas liquid chromatography-mass spectrometry showed the bile acid metabolite to be 12a-hydroxy-3-oxo-4cholenoic acid. Based on these results the proposed structure of this novel bile acid nucleotide is shown in Structure 1. Results of studies with 32P-labeled bile acid nucleotide suggest a rapid loss of phosphate from the bile acid when treated with nucleotide pyropho~phatase.~ We believe that the bile acid nucleotide described in this study is formed by 7a-dehydroxylase and is an intermediate in the 7-dehydroxylation reaction. These results suggest a new mechanism of bile acid ?'-dehydroxylation. The mechanism of 7a-dehydroxylation of bile acid was first investigated by Samuelsson (15) using doubly labeled cholic acid fed to conventional rats. The reaction was proposed to occur by a diaxial trans elimination of water yielding a postulated A6steroid intermediate followed by trans hydrogenation at the 6a and 7@ positions. Investigations by Ferrari et al. (16) and White et al. (6) supported this mechanism by showing the reduction of chemically synthesized 3a,12a-dihydroxy-5@chol-6-enoic acid to deoxycholic acid by cell extracts of Clostridium bifermentans and Eubucterium sp. V.P.I. 12708, respectively. However, both 7a,7@-dehydroxylase and A6-steroid 108.9 * 1.9 112.9 * 3.9 lW.0 * 2.0