Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708

Abstract

Southern blot analysis indicated that the gene encoding the constitutive, NADP-linked bile acid 7a-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708 was located on a 6.5-kb EcoRI fragment of the chromosomal DNA. This fragment was cloned into bacteriophage lambda gtll, and a 2.9-kb piece of this insert was subcloned into pUC19, yielding the recombinant plasmid pBH51. DNA sequence analysis of the 7a-hydroxysteroid dehydrogenase gene in pBH51 revealed a 798-bp open reading frame, coding for a protein with a calculated molecular weight of 28,500. A putative promoter sequence and ribosome binding site were identified. The 7a-hydroxysteroid dehydrogenase mRNA transcript in Eubacterium sp. strain VPI 12708 was about 0.94 kb in length, suggesting that it is monocistronic. An Escherichia coli DH5a transformant harboring pBH51 had approximately 30-fold greater levels of 7a-hydroxysteroid dehydrogenase mRNA, immunoreactive protein, and specific activity than Eubacterium sp. strain VPI 12708. The 7af-hydroxysteroid dehydrogenase purified from the pBH51 transformant was similar in subunit molecular weight, specific activity, and kinetic properties to that from Eubacterium sp. strain VPI 12708, and it reacted with antiserum raised against the authentic enzyme on Western immunoblots. Alignment of the amino acid sequence of the 7a-hydroxysteroid dehydrogenase with those of 10 other pyridine nucleotide-linked alcohol/polyol dehydrogenases revealed six conserved amino acid residues in the N-terminal regions thought to function in coenzyme binding.Many members of the human intestinal microflora can modify the glycine and taurine conjugates of the primary bile acids, cholic acid and chenodeoxycholic acid, producing up to 20 different bile acid metabolites (21). The predominant modifications include deconjugation of the glycine and taurine moieties by bile salt hydrolase, removal of 7-hydroxy groups (7-dehydroxylation), and dehydrogenation of a-and ,B-hydroxy groups at carbons 3, 7, and 12 by stereospecific bile acid hydroxysteroid dehydrogenases to yield oxo bile acids.Quantitatively, the most important of these transformations is the 7ux-dehydroxylation of cholic and chenodeoxycholic acids, producing deoxycholic and lithocholic acids, respectively (43). The human intestinal anaerobic bacterium, Eubacterium sp. strain VPI 12708, has a multistep 7a-dehydroxylation pathway which is induced by addition of cholic acid to the culture medium (6,9,11, 22,(46)(47)(48). Several bile acid-inducible (bai) genes encoding proteins involved in this pathway have been cloned and sequenced (10,11,18,31,49,50).7a-Hydroxysteroid dehydrogenase is the most common class of bile acid hydroxysteroid dehydrogenases found in nature (3), having been detected in numerous genera of bacteria (3, 14-16, 28, 30, 37) and in mammalian liver (1). 7a-Hydroxysteroid dehydrogenases have been purified from Bacteroides species (23,29,41), Escherichia coli (28,36,37), and rat liver microsomes (1). Recently, two 7a-hydroxysteroid dehydrogenases have been pu...

Bile salt biotransformations by human intestinal bacteria

“…Regulation of 7a/b-HSDH expression is generally growth phase-dependent and inducible by bile acid substrates (Table 2). C. scindens and E. coli constitutively express 7a-HSDHs and are noninducible (69,85). Unexpectedly, the nonsubstrate DCA can induce 7a-HSDH expression in C. absonum and C. sordellii, although the reason for this induction remains unclear (70,76).…”

“…7a/b-HSDHs are widespread among the bacteroides and clostridia as well as in E. coli and Ruminococcus species (Table 2) (54,64,(69)(70)(71)(72)(73)(74). In addition, several intestinal clostridia express both 7a-and 7b-HSDHs and have been shown to epimerize the 7a/b-hydroxy group (75,(76)(77)(78).…”

Assignment of Eubacterium sp. VPI 12708 and related strains with high bile acid 7alpha-dehydroxylating activity to Clostridium scindens and proposal of Clostridium hylemonae sp. nov., isolated from human faeces.

“…After the characterization, many studies (Baron et al, 1991 ;Coleman et al, 1987 ;Doerner et al, 1997 ;Franklund et al, 1993 ;Mallonee & Hylemon, 1996 ;Mallonee et al, 1990Mallonee et al, , 1992 on strain VPI 12708, especially on the bai (bile acid inducible) operon, have been reported. However, detailed studies concerning taxonomic research on strain VPI 12708 have not appeared.…”

Characterization and regulation of the NADP-linked 7 alpha-hydroxysteroid dehydrogenase gene from Clostridium sordellii

“…The majority of those bacterial steroid and bile acid dehydrogenases which have been characterized at the sequence level are related as members of the short-chain alcohol dehydrogenase family (8,49,60). As additional enzymes active on a variety of substrates are identified as members of this family, investigators will have available a unique data set which can be used for studies on the role of specific residues and domains on enzyme conformation, substrate specificity, and reaction stereospecificity.…”

“…In organisms such as E. coli and a Eubacterium sp., for example, the 7at-hydroxysteroid dehydrogenase (7xt-HSDH) is expressed constitutively (8,19,40), while two 3ot-HSDHs from the Eubacterium sp. are inducible by bile acids containing a 7ot-hydroxy group (13,23,58).…”