Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme

Yoshimoto, Tadashi; Higashi, Hideaki; Kanatani, Akio; Lin, Xiong-Shui; Nagai, Hirokazu; Oyama, Hiroshi; Kurazono, K; Tsuru, Daisuke

doi:10.1128/jb.173.7.2173-2179.1991

Cited by 95 publications

(71 citation statements)

References 23 publications

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“…Phage lysates prepared from nonrecombinant lambda gtll or cell extracts of E. coli DH5a(pUC19) had no detectable NADP-dependent 7a-hydroxysteroid dehydrogenase activity (Table 1). Although certain strains of E. coli possess an NAD-dependent 7oc-hydroxysteroid dehydrogenase (19,28,37,52), the NAD-dependent 7a-hydroxysteroid dehydrogenase activities in cell extracts of E. coli DH5ot(pUC19) or DH5ot(pBH51) ( Table 1) were about 1,000-and 500-fold less, respectively, than that reported for cell extracts of E. coli 080 (338 U/mg of protein) (37). Moreover, all of the NADdependent activities reported in Table 1 were substantially less than the corresponding NADP-dependent activities, and much higher concentrations of cholic acid and NAD were required to detect the former.…”

Section: Resultsmentioning

confidence: 99%

“…This study describes the cloning, sequencing, and expression of the gene encoding this enzyme. The gene encoding NAD-linked 7a-hydroxysteroid dehydrogenase from E. coli HB101 has also been cloned, sequenced, and expressed in E. coli DH1, and the enzyme has been purified and characterized (52). The Eubacterium and E. coli 7ot-hydroxysteroid dehydrogenases have comparable native molecular weights (124,000 and 120,000, respectively) and subunit apparent molecular weights (32,000 and 28,000, respectively), are synthesized constitutively (i.e., in the presence or absence of bile acids), can use free or conjugated 7a-hydroxy bile acids as substrates (16,52), and are unaffected by metal ion chelators.…”

Section: Discussionmentioning

confidence: 99%

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

1991

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

1991

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“…1), and unconjugated bile acids should be able to penetrate, albeit slowly, through the lipid bilayer domain of this membrane (see reference 23). Since these unconjugated bile acids exist largely in uncharged protonated forms at the acidic pH of the periplasm, they should cross the lipid bilayer domain of the cytoplasmic membrane easily, and the cloning from E. coli of a 7␣-hydroxysteroid dehydrogenase gene, whose product appears to have no signal sequence (32), suggests that the substrates of this enzyme, bile acids, do reach the cytoplasm. In the presence of a constant influx of bile acids from the environment, an active efflux mechanism would be needed to avoid the toxic, detergent action of these compounds, in addition to their possible detoxification through metabolism.…”

Section: Discussionmentioning

confidence: 99%

Active efflux of bile salts by Escherichia coli

1997

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Enteric bacteria such as Escherichia coli must tolerate high levels of bile salts, powerful detergents that disrupt biological membranes. The outer membrane barrier of gram-negative bacteria plays an important role in this resistance, but ultimately it can only retard the influx of bile salts. We therefore examined whether E. coli possessed an energy-dependent efflux mechanism for these compounds. Intact cells of E. coli K-12 appeared to pump out chenodeoxycholate, since its intracellular accumulation increased more than twofold upon deenergization of the cytoplasmic membrane by a proton conductor. Growth inhibition by bile salts and accumulation levels of chenodeoxycholate increased when mutations inactivating the acrAB and emrAB gene clusters were introduced. The AcrAB system especially appeared to play a significant role in bile acid efflux. However, another efflux system(s) also plays an important role, since the accumulation level of chenodeoxycholate increased strongly upon deenergization of acrA emrB double mutant cells. Everted membrane vesicles accumulated taurocholate in an energy-dependent manner, apparently consuming ⌬pH without affecting ⌬⌿.The efflux thus appears to be catalyzed by a proton antiporter. Accumulation by the everted membrane vesicles was not decreased by mutations in acr and emrB genes and presumably reflects activity of the unknown system seen in intact cells. It followed saturation kinetics with V max and K m values in the neighborhood of 0.3 nmol min ؊1 mg of protein ؊1 and 50 M, respectively.

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“…(45,46). The 7␣-hydroxysteroid dehydrogenase (7␣-HSDH), catalyzes the NAD ϩ -dependent dehydrogenation of the hydroxyl group at position seven on the steroid skeleton of bile acids (52). Amino acid sequence alignment of M. tuberculosis InhA with E. coli 7␣-HSDH shows 22% identity, and superposition of their crystal structures shows that they share a very similar backbone topology (1.5-Å r.m.s.…”

Section: Inha-nadmentioning

confidence: 99%

Crystal Structure of the Mycobacterium tuberculosis Enoyl-ACP Reductase, InhA, in Complex with NAD+ and a C16 Fatty Acyl Substrate

Rozwarski¹,

Vilchèze²,

Sugantino³

et al. 1999

Journal of Biological Chemistry

262

281

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Mammals, birds, and yeast produce fatty acids via a FAS-I system, a biosynthetic pathway in which each enzymatic activity resides on the single polypeptide chain of a very large multifunctional enzyme (1-3). In contrast, most plants and bacteria utilize a FAS-II system in which each of the enzymatic activities corresponds to individual polypeptides (4 -6). The best characterized FAS-II system is that of Escherichia coli, which includes ␤-ketoacyl-ACP synthases (FabB, FabF, and FabH), a ␤-ketoacyl-ACP reductase (FabG), ␤-hydroxyacyl-ACP dehydrases (FabA and FabZ), and an enoyl-ACP reductase (currently known as FabI and formerly known as EnvM). The E. coli FAS-II system initializes fatty acid biosynthesis by condensing acetyl and malonyl substrates, and then it continues the process by elongating the resulting product through the consecutive addition of C2 units, until the fatty acyl chain reaches a length of about 16 carbons.Some bacteria, such as mycobacteria, possess both a FAS-I and a FAS-II system (7). The mycobacterial FAS-I system displays a bimodal distribution of products centered on C16 and C24 -C26 (7,8). This FAS-II system prefers C16 as a starting substrate (7) and can extend up to C56 (9), indicating that the mycobacterial FAS-II system utilizes the products of the FAS-I system as primers to extend fatty acyl chain lengths even further. The longer chain products of the FAS-II system are the precursors of mycolic acids. Mycolic acids are long chain ␣-alkyl-␤-hydroxy fatty acids, which are a major component of mycobacterial cell walls (10, 11).Isoniazid, a drug used as a first-line antibiotic for the treatment of Mycobacterium tuberculosis infections for the past 40 years, is known to inhibit mycolic acid biosynthesis (12-15). More recently, isoniazid was shown to prevent radiolabel from being incorporated into the chain extension of C24 -C26 fatty acyl substrates (16 -20), suggesting that the target of isoniazid action is within the mycobacterial FAS-II system. Using a genetic approach to isolate the isoniazid target, a single open reading frame was identified (referred to as inhA) in which transformation of the wild-type gene carried on a multicopy plasmid or allelic exchange with a single amino acid substitution mutant (Ser 94 to Ala) was sufficient to confer isoniazid resistance in Mycobacterium smegmatis (21).The protein encoded by the inhA gene, referred to as InhA, has a similar amino acid sequence to two previously characterized enoyl-ACP reductases, namely FabI from E. coli (28% identity) (22)(23)(24)(25), and ENR 1 from Brassica napus (oilseed rape) (23% identity) (26,27). Further analysis revealed that InhA catalyzes the NADH-dependent reduction of the trans double bond between positions C2 and C3 of fatty acyl substrates (28). In addition, InhA prefers fatty acyl substrates of C16 or greater, consistent with its being a member of the mycobacterial FAS-II system (28).The connection between isoniazid action and InhA inhibition is somewhat complicated. Isoniazid is a pro-drug that must be conv...

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Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme

Cited by 95 publications

References 23 publications

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

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

Active efflux of bile salts by Escherichia coli

Crystal Structure of the Mycobacterium tuberculosis Enoyl-ACP Reductase, InhA, in Complex with NAD+ and a C16 Fatty Acyl Substrate

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