Clostridium scindens baiCD and baiH genes encode stereo-specific 7alpha/7beta-hydroxy-3-oxo-delta4-cholenoic acid oxidoreductases.
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. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7alpha-hydroxy-3-oxo-Delta4-cholenoic acid oxidoreductase.
Kang
Dae-Joong
DJ
Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA.
Ridlon
Jason M
JM
Moore
Doyle Ray
DR
Barnes
Stephen
S
Hylemon
Phillip B
PB
D000595:Amino Acid Sequence; D002264:Carboxylic Acids / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D002855:Chromatography, Thin Layer; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D017124:Conserved Sequence; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity
D002264:Carboxylic Acids; D011994:Recombinant Proteins; D010088:Oxidoreductases