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. 2013 Nov;54(11):3062-9.
doi: 10.1194/jlr.M039834. Epub 2013 Jun 1.

Contribution of the 7β-hydroxysteroid dehydrogenase from Ruminococcus gnavus N53 to ursodeoxycholic acid formation in the human colon (VSports注册入口)

Ja-Young Lee  1 , Hisashi Arai, Yusuke Nakamura, Satoru Fukiya, Masaru Wada, Atsushi Yokota
Affiliations

Contribution of the 7β-hydroxysteroid dehydrogenase from Ruminococcus gnavus N53 to ursodeoxycholic acid formation in the human colon (VSports最新版本)

Ja-Young Lee et al. J Lipid Res. 2013 Nov.

V体育安卓版 - Abstract

Bile acid composition in the colon is determined by bile acid flow in the intestines, the population of bile acid-converting bacteria, and the properties of the responsible bacterial enzymes. Ursodeoxycholic acid (UDCA) is regarded as a chemopreventive beneficial bile acid due to its low hydrophobicity. However, it is a minor constituent of human bile acids. Here, we characterized an UDCA-producing bacterium, N53, isolated from human feces. 16S rDNA sequence analysis identified this isolate as Ruminococcus gnavus, a novel UDCA-producer. The forward reaction that produces UDCA from 7-oxo-lithocholic acid was observed to have a growth-dependent conversion rate of 90-100% after culture in GAM broth containing 1 mM 7-oxo-lithocholic acid, while the reverse reaction was undetectable. The gene encoding 7β-hydroxysteroid dehydrogenase (7β-HSDH), which facilitates the UDCA-producing reaction, was cloned and overexpressed in Escherichia coli. Characterization of the purified 7β-HSDH revealed that the kcat/Km value was about 55-fold higher for the forward reaction than for the reverse reaction, indicating that the enzyme favors the UDCA-producing reaction. As R. gnavus is a common, core bacterium of the human gut microbiota, these results suggest that this bacterium plays a pivotal role in UDCA formation in the colon. VSports手机版.

Keywords: bile acid conversion; epimerization; intestinal bacteria; secondary bile acids V体育安卓版. .

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Figures

Fig. 1.
Fig. 1.
Epimerization reaction from CDCA to UDCA, which is catalyzed by the 7α-HSDH and 7β-HSDH enzymes. The 7β-HSDH from R. gnavus N53 preferentially catalyzes the UDCA-forming reaction (reductive reaction) as compared with the 7-oxo-LCA-forming reaction (oxidative reaction), which is denoted by the thickness of the arrow lines.
Fig. 2.
Fig. 2.
TLC analysis of biotransformation by R. gnavus N53. The bacterial strain was cultured anaerobically in GAM broth with 0.1 mM 7-oxo-LCA for 48 h (lane 1). Lanes 2, 3, and 4 are authentic, CDCA, UDCA, and 7-oxo-LCA, respectively.
Fig. 3.
Fig. 3.
Time course of UDCA formation from 7-oxo-LCA by three bacterial strains: (A) R. gnavus N53, (B) C. aerofaciens ATCC 25986T, and (C) R. gnavus ATCC 29149T. Bacteria were cultured anaerobically in GAM broth supplemented with 1 mM 7-oxo-LCA. Data are the means of at least three independent experiments. Bars represent the means ± SD. Dashed line, growth expressed as optical density at 660 nm (OD660); open square, residual 7-oxo-LCA in the culture broth; open circle, UDCA in the culture broth formed from 7-oxo-LCA.
Fig. 4.
Fig. 4.
Amino acid sequence alignment of 7β-HSDH from R. gnavus N53 and selected other HSDH proteins, including those from R. gnavus ATCC 29149T (GenBank accession number, ZP_02041813; not shown as it has the identical amino acid sequence to that from R. gnavus N53), C. aerofaciens ATCC 25986T (GenBank, ZP_01773061), a putative short-chain dehydrogenase from R. torques L2-14 (GenBank, CBL26204), and 7β-HSDH from Cl. absonum DSM 599T (GenBank, AET80684). Residues inside the box indicate the N-terminal cofactor-binding site. Catalytic triad residues are indicated by arrows.
Fig. 5.
Fig. 5.
SDS-PAGE. M, marker; Lane 1, crude enzyme; Lane 2, purified recombinant protein (indicated by the arrow). The marker standards were phosphorylase (97,400 Da), BSA (66,300 Da), aldolase (42,400 Da), carbonic anhydrase (30,000 Da), and trypsin inhibitor (20,100 Da).
Fig. 6.
Fig. 6.
Reversibility of the 7β-HSDH reaction as assessed using purified enzyme. HPLC chromatogram of the mixture for the (A) reductive reaction and (B) oxidative reaction. The reductive reaction mixture contained 200 μM sodium 7-oxo-LCA, 400 μM NADPH, 50 μl (0.6 μmol∙min−1∙ml -1) enzyme, and 100 mM MES (pH 6.0) in a final volume of 0.5 ml. The oxidative reaction mixture contained 1 mM sodium UDCA, 1 mM NADP+, 50 μl enzyme, and 100 mM glycine-NaOH (pH 10.0). Reaction mixtures without the enzyme served as controls. All reaction mixtures, after incubation at 37°C for 1 h, were analyzed by HPLC as described previously (32). Solid line, reaction in the presence of 7β-HSDH; dashed line, control reaction mixture without 7β-HSDH. Arrows indicate the conversion product of each reaction.
Fig. 7.
Fig. 7.
Effect of pH on the activity of 7β-HSDH from R. gnavus N53 in (A) reductive and (B) oxidative reactions. The activity of 7β-HSDH was assayed at a pH range from 5.0 to 11.5 using the following buffers, each at 100 mM and 37°C: sodium acetate (filled square); MES (filled circle); Tris-HCl (filled triangle); glycine-NaOH (filled diamond); and N-tris[hydroxymethyl]methyl-4-aminobutane-sulfonic acid (TABS) (cross). *micromoles of NADP(H) per minute per milligram of protein.
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