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. 2008 Jan-Feb;1781(1-2):16-25.
doi: 10.1016/j.bbalip.2007.10.008. Epub 2007 Nov 7.

"VSports app下载" Clostridium scindens baiCD and baiH genes encode stereo-specific 7alpha/7beta-hydroxy-3-oxo-delta4-cholenoic acid oxidoreductases

Dae-Joong Kang  1 , Jason M Ridlon, Doyle Ray Moore 2nd, Stephen Barnes, Phillip B Hylemon
Affiliations

Clostridium scindens baiCD and baiH genes encode stereo-specific 7alpha/7beta-hydroxy-3-oxo-delta4-cholenoic acid oxidoreductases

Dae-Joong Kang et al. Biochim Biophys Acta. 2008 Jan-Feb.

Abstract

Secondary bile acids, formed by intestinal bacteria, are suggested to play a significant role in cancers of the gastrointestinal tract in humans. Bile acid 7alpha/beta-dehydroxylation is carried out by a few species of intestinal clostridia which harbor a multi-gene bile acid inducible (bai) operon. Several genes encoding enzymes in this pathway have been cloned and characterized. However, no gene product(s) has yet been assigned to the production of 3-oxo-Delta4-cholenoic acid intermediates of cholic acid (CA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). We previously reported that the baiH gene encodes an NADH:flavin oxidoreductase (NADH:FOR); however, the role of this protein in bile acid 7-dehydroxylation is unclear. Homology searches and secondary structural alignments suggest this protein to be similar to flavoproteins which reduce alpha/beta-unsaturated carbonyl compounds. The baiH gene product was expressed in Escherichia coli, purified and discovered to be a stereo-specific NAD(H)-dependent 7beta-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase. Additionally, high sequence similarity between the baiH and baiCD gene products suggests the baiCD gene may encode a 3-oxo-Delta4-cholenoic acid oxidoreductase specific for CDCA and CA. We tested this hypothesis using cell extracts prepared from E VSports手机版. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7alpha-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase. .

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Figures

Figure 1
Figure 1. SDS-PAGE and immunoblot analysis of baiH gene product (NADH:FOR) overexpression and purification
(a.) Proteins were separated on a 10 % SDS-polyacrylamide gel and stained with Coomassie Blue R-250. Lane 1, cell free extract after IPTG induction (20 μg loaded); Lane 2, flow-through from DEAE 40HR (20 μg loaded); Lane 3, flow-through from Cibacron Blue 3GA agarose (20 μg loaded); lane 4, flow-through from Hydroxyapatite CHT5-1 column (5 μg of purified enzyme loaded). (b.) Immunoblot of SDS-PAGE using anti-BaiH antiserum.
Figure 1
Figure 1. SDS-PAGE and immunoblot analysis of baiH gene product (NADH:FOR) overexpression and purification
(a.) Proteins were separated on a 10 % SDS-polyacrylamide gel and stained with Coomassie Blue R-250. Lane 1, cell free extract after IPTG induction (20 μg loaded); Lane 2, flow-through from DEAE 40HR (20 μg loaded); Lane 3, flow-through from Cibacron Blue 3GA agarose (20 μg loaded); lane 4, flow-through from Hydroxyapatite CHT5-1 column (5 μg of purified enzyme loaded). (b.) Immunoblot of SDS-PAGE using anti-BaiH antiserum.
Figure 2
Figure 2. TLC autoradiograph of BaiCD and BaiH catalyzed NAD(H)-dependent 3-oxo-Δ4-cholenoic acid oxidoreductase activity assays
The reactions were carried out in a 500 μl volume consisting of 50 mM sodium phosphate buffer (pH 6.8) containing 225 μM of each respective substrate with a specific activity of 0.01 μCi μmole-1. A: [24-14C] 3-dehydro-CDCA standard, B: [24-14C] 3-dehydro-UDCA, C: [24-14C] 3-dehydro-CDCA + 100 μg cell-free extract of E. coli (plasmid, no insert) D: [24-14C] 3-dehydro-CDCA + 1.6 U purified BaiH, E: [24-14C] 3-dehydro-CDCA + 100 μg E.coli BL21(DE3) expressing baiCD. F: [24-14C] 3-dehydro-UDCA + 100 μg E. coli BL21(DE3) (plasmid, no insert), G: [24-14C] 3-dehydro-UDCA + 100 μg cell-free extract of E.coli BL21(DE3) expressing baiCD, H: [24-14C] 3-dehydro-UDCA + 1.6 U purified BaiH. All reactions were performed with 1.5 mM NAD+ as co-substrate.
Figure 3
Figure 3. SDS-PAGE of cell-extract of recombinant E. coli BL21(DE3) overexpressing baiCD gene
(M) Marker (UI) uninduced, (I) induced 1 mM IPTG. 30 μg protein loaded and gel stained with Coomassie brilliant blue R-250. A large band at ~ 70 kDa was detected in IPTG induced cell-extracts indicating overexpression of baiCD gene.
Figure 4
Figure 4. Amino acid sequence alignment of the BaiH (AAC45417) from C. scindens VPI 12708 with protein homologues
2,4-dienoyl-CoA reductase (dcr) (AAC76116) from E. coli, 2-enoate reductase (CAA71086) from Clostridium tyrobutyricum, the baiCD gene product from Clostridium scindens (EF539210) and Clostridium hiranonas (AAF22846) and the baiH gene product from C. hiranonis (EF539211) Secondary structural elements based on the crystal structure of DCR (21) are shown (below alignment). Predicted secondary structural elements of the BaiH protein are also shown (above alignment). Arrows correspond to beta-sheets and loops to alpha helices. Residues corresponding to FMN binding (■), FAD binding (*), and cysteine residues involved in a 4Fe-4S center (♦) from the crystal structure of DCR are also shown. The alignment was generated with the T-COFFEE program (42) and sequence similarities/identities highlighted with Boxshade 3.1 (http://www.ch.embnet.org/software/BOX_form.html).
Figure 5
Figure 5. Proposed reactions catalyzed by the baiCD and baiH gene product
R group may be the carboxylic acid or the coenzyme A conjugate
See this image and copyright information in PMC

References

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