Nucleotide sequence and regulation of a gene involved in bile acid 7-dehydroxylation by Eubacterium sp. strain VPI 12708

Abstract

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium that has inducible bile acid 7-dehydroxylation activity. At least four new polypeptides were synthesized after addition of primary bile acids to the growth medium. One of these, of molecular weight 27,000 (P-27), was shown to be involved in the 7-dehydroxylation reaction sequence. The gene coding for P-27 was cloned, and the entire DNA sequence for the protein-coding region was determined. In addition, sequence information was obtained for 294 bases upstream from the translational start codon and 329 bases downstream from the stop codon. Induction studies with a synthetic oligonucleotide probe (16-mer) revealed the presence of a cholic acid-inducible mRNA species approximately 900 bases long. A 5' terminus of this mRNA was detected by primer extension analysis, and the location of the 3' terminus of the mRNA was estimated by using Si nuclease mapping. The 3' terminus of the mRNA contained a large element with dyad symmetry of unknown function. The open reading frame contained 249 codons, and the corresponding polypeptide had a calculated molecular weight of 26,745. The amino acid sequence of P-27 showed significant homology to several previously described alcohol-polyol dehydrogenases ("nonzinc" dehydrogenases), especially in the region believed to contain a pyridine nucleotide-binding domain. The implications of this homology and the possible function of P-27 in bile acid 7-dehydroxylation are discussed.

Prokaryotic 20β‐hydroxysteroid dehydrogenase is an enzyme of the ‘short‐chain, non‐metalloenzyme’ alcohol dehydrogenase type

“…Consequently, it may be concluded that the 'short-chain dehydrogenase family' includes not only insect alcohol dehydrogenase [2] and prokaryotic dehydrogenases of sugar metabolism [4], but also prokaryotic steroid dehydrogenases. This is compatible with the further inclusion of two other proteins involved in prokaryotic dehydroxylation [5,6] and suggests that they are also hydroxysteroid dehydrogenases. The results enlarge the short-chain alcohol dehydrogenase family and establish it to include many different enzyme specificities.…”

“…The classical liver and yeast alcohol dehydrogenases belong to the latter family, the insect enzyme to the former, and was long ago found to be different [3]. Divergent members of the 'short-chain dehydrogenase type' have been characterized and additional sugar, steroid and other dehydrogenases were added to the family [2,[4][5][6][7], as well as species variants for the Drosophila alcohol dehydrogenase [8]. The enzymes initially characterized were non-mammalian, but recently mammalian proteins have also been found to belong to this group, thus linking a human prostaglandin [9] and a human steroid dehydrogenase [10,11] to the prokaryotic/insect short-chain dehydrogenase type.…”

The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase

“…DISCUSSION It has been previously proposed that the formation of a bile acid-adenosine nucleotide is the initial step in the bile acid 7a-dehydroxylation pathway in Iiubacterium sp. strain VPI 12708 (4,6). In this article, we report that a bile acid-CoA ligation activity is encoded by the baiB gene from Eubacterium sp.…”

“…The 7a-dehydroxylation activity has been proposed to proceed by a multistep pathway where the bile acid is first linked to an adenosine nucleotide upon entering the cell (4,6). The bile acid then undergoes a pair of sequential oxidation reactions followed by loss of the 7ao-hydroxyl group in a dehydration step, and finally the 3-oxo-_A46-steroid intermediate undergoes a three-step reduction, yielding the 7-dehydroxylated product.…”

“…This bacterium synthesizes several new polypeptides after cholic acid induction (24,36). Three of these inducible polypeptides have been purified, and the genes encoding them have been cloned and sequenced (5,6,20,38). One of the purified polypeptides, a 27,000-Mr species, is encoded by three separate genes (baiAl, baiA42, and baiA3) (5,6,13,39).…”