Expression in Escherichia coli and characterization of a bile acid-inducible 3 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.
We have previously cloned and sequenced three members of a bile acid-inducible gene family from Eubacterium sp. strain VPI 12708 that encode 27,000-M(r) polypeptides. Two copies of these genes (baiA1 and baiA3) are identical, while the third copy (baiA2) encodes a polypeptide sharing 92% amino acid identity with the baiA1 and baiA3 gene products. We have overexpressed the baiA1 gene in Escherichia coli and analyzed the expressed activity. Thin-layer chromatography of 14C-labeled bile acid products from reactions using cell-free extracts revealed a 3 alpha-hydroxysteroid dehydrogenase activity for the BaiA1 protein. The BaiA1 protein could utilize both NAD+ and NADP+, and the preferred steroid substrate was the cholyl-coenzyme A conjugate rather than free cholic acid. These results show that the BaiA proteins are novel 3 alpha-hydroxysteroid dehydrogenases.
Mallonee
D H
DH
Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0678, USA.
Lijewski
M A
MA
Hylemon
P B
PB
D015096:3-Hydroxysteroid Dehydrogenases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000595:Amino Acid Sequence; D001647:Bile Acids and Salts / Q000378:metabolism / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D005051:Eubacterium / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D001647:Bile Acids and Salts; D009243:NAD; D009249:NADP; D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)