Behavior of 3α- and 7α-hydroxysteroid dehydrogenases on chenodeoxycholate substituted sepharose

Chenodeoxycholate (3α-, 7α-dihydroxy-5β-cholanoate) was linked to Sepharose 4B by an ethylenediamine bridge. When 3α-hydroxysteroid dehydrogenase and 7α-hydroxysteroid dehydrogenase preparations were applied to a column of covalently linked Chenodeoxycholate, both enzymes were retarded at pH 6.7; the 7α-OH oriented enzyme more than the 3α-OH enzyme. Approximately forty-fold purification of 7a-hydroxysteroid dehydrogenase was achieved in one step. Although no significant purification of 3α-hydroxysteroid dehydrogenase occurred, the background value in the fluorometric enzymatic estimation of bile acids by eluted 3α-hydroxysteroid dehydrogenase was markedly reduced. Molecular weight estimation by Sephadex G-200 gave the values of 47,000 for 3α-hydroxysteroid dehydrogenase and 105,000 for 7α-hydroxy-steroid dehydrogenase.     

Simple enzymatic detection method for urinary sulfated 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis

Urinary sulfated primary bile acids, 7α-hydroxy bile acids, are detected by an enzymatic method using 7α-hydroxysteroid dehydrogenase (EC 1.1.1.-, 7α-HSD) after chromatographic fractionation on Sephadex G-25. Urinary sulfated or glucuronated bile acids are hydrolyzed by β-glucuronidase/sulfatase (EC 3.2.1.31/EC 3.1.6.1) from Helix pomatia and then released 7α-hydroxy bile acids are detected with 7α-HSD in the presence of β-NAD⁺, diaphorase (EC 1.6.99.2, from Clostridium kluyveri) and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride. The absorbance of formazan formed during the enzymic reaction is measured at 500 nm. Excretion values of 7α-hydroxy bile acids in normal subjects and in patients with acute hepatitis were compared. This enzymatic detection method for the excretion pattern of urinary 7α-hydroxy bile acids may be useful for clinical diagnosis.     

Continuous-flow automated assay of steroids with nylon-tube-immobilized hydroxysteroid dehydrogenases

Several NAD(P)⁺-dependent hydroxysteroid dehydrogenases, namely 3α-hydroxysteroid dehydrogenase, β-hydroxysteroid dehydrogenase, 7α-hydroxysteroid dehydrogenase, and 12α-hydroxysteroid dehydrogenase were separately immobilized on nylon tubes for the continuous-flow automated assay of hydroxysteroids. 3α-Hydroxysteroid dehydrogenase was also immobilized on pore glass. Spectrophotometric monitoring in the visible region, where blank values were markedly reduced, was achieved through the Meldola blue catalyzed transfer of hydrogen from NAD(P)H to a tetrazolium salt. Nylon-tube-immobilized enzymes maintained 45–55% of the original activity after 1 month of intermittent use. The operational range, using the “end point” approach, was 1–25 nmol of steroid and the assay speed 10–15 samples/h. Reliable results were obtained in the determination of 3α-hydroxysteroids and 3β,17β-hydroxysteroids in urine and total bile acids in serum.     

The catalytic promiscuity of a microbial 7α-hydroxysteroid dehydrogenase. Reduction of non-steroidal carbonyl compounds

A thermostable 7α-hydroxysteroid dehydrogenase from Bacteroides fragilis ATCC 25285 was found to catalyze the reduction of various benzaldehyde analogues to their corresponding benzyl alcohols. The enzyme activity was dependent upon the substituent on the benzene ring of the substrates. Benzaldehydes with electron-withdrawing substituent usually showed higher activity than those with electron-donating groups. Furthermore, this enzyme was tolerant to some organic solvents. These results together with previous studies suggested that 7α-hydroxysteroid dehydrogenase from B. fragilis might play multiple functional roles in biosynthesis and metabolism of bile acids, and in the detoxification of xenobiotics containing carbonyl groups in the large intestine. In addition, its broad substrate spectrum offers great potential for finding applications not only in the synthesis of steroidal compounds of pharmaceutical importance, but also for the production of other high-value fine chemicals.     

Rapidly directional biotransformation of tauroursodeoxycholic acid through engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bear bile powder is a precious medicinal material. It is characterized by high content of tauroursodeoxycholic acid (TUDCA) at a ratio of 1.0–1.5 to taurochenodeoxycholic acid (TCDCA). Here, we reported the biotransformation of tauroursodeoxycholic acid (TUDCA) through Escherichia coli engineered with a two-step mimic biosynthetic pathway of TUDCA from taurochenodeoxycholic acid (TCDCA). Two 7α-hydroxysteroid dehydrogenase (7α-HSDH) and two 7β-hydroxysteroid dehydrogenase (7β-HSDH) genes (named as α₁, α₂, β₁, and β₂) were selected and synthesized to create four pathway variants using ePathBrick. All could convert TCDCA to TUDCA and the one harboring α₁ and β₂ (pα₁β₂) showed the strongest capability. Utilizing the oxidative and reductive properties of 7α- and 7β-HSDH, an ideal balance between TUDCA and TCDCA was established by optimizing the fermentation conditions. By applying the optimal condition, E. coli containing pα₁β₂ (BL-pα₁β₂) produced up to 1.61 ± 0.13 g/L of TUDCA from 3.23 g/L of TCDCA at a ratio of 1.3 to TCDCA. This study provides a potential approach for bear bile substitute production from cheap and readily available chicken bile.     

Problems in the measurement of bile acids with 3 -hydroxysteroid dehydrogenase

Difficulties encountered in the analysis of bile acids with a crude bacterial 3α-hydroxysteroid dehydrogenase were found to result from the use of alcohol which caused generation of NADH. A microanalytical technique was developed without alcohol which was suitable for the measurement of 0.01 μmoles of bile salt.     

An enzymatic cycling method for the determination of serum total bile acids with recombinant 3α-hydroxysteroid dehydrogenase

A highly sensitive enzymatic cycling method was developed for the serum total bile acids assay. We constructed a prokaryotic expression system to prepare the recombinant 3α-hydroxysteroid dehydrogenase in place of the natural enzyme and for the first time used it in the total bile acids assay. The production rate of thio-NADH correlated with the bile acids concentration and was measured by the change of absorbance at 405/660 nm. The enzymatic cycling method could detect 0.22 μmol/L total bile acids in serum. Within-run and between-run imprecisions were 1.2-3.7% and 2.3-4.8%, respectively. The calibration curve for total bile acids in serum was linear between 0.5 and 180 μmol/L. This method was free from interference by bilirubin, hemoglobin, ascorbate, and lactate dehydrogenase. In conclusion, serum total bile acids could be measured by the enzymatic cycling method with recombinant 3α-hydroxysteroid dehydrogenase as the tool enzyme.     

Purification and characterization of NADP-dependent 7β-hydroxysteroid dehydrogenase from Peptostreptococcus productus strain b-52

An NADP-dependent 7β-hydroxysteroid dehydrogenase was purified 11.5-fold over the activity in crude cell extracts prepared from Peptostreptococcus productus strain b-52, by using Sephadex G-200 and DEAE-cellulose column chromatography. 7β-Dehydrogenation was the sole transformation of bile acids catalyzed by the partially purified enzyme. The enzyme preparation (spec. act. 2.781 IU per mg protein) had an optimum pH of 9.8. Lineweaver-Burk plots showed a Michaelis constant (K_m) value of 0.05 mM for 3α,7β-dihydroxy-5β-cholanic acid whereas higher values were obtained with 3α,7β-dihydroxy-5β-cholanoyl glycine (0.20 mM), and 3α,7β-dihydroxy-5β-cholanoyl taurine (0.26 mM). NADP but not NAD could function as an electron acceptor, and has a K_m value of 0.30 mM. A molecular weight of 64 000 was determined by SDS-polyacrylamide gel electrophoresis. The addition of 0.4 mM of either bile acid to the growth medium suppressed not only cell growth, but also the enzyme yield.     

Deconjugation of bile salts by Bacteroids and Clostridium

Deconjugation of bile salts by four strains of Bacteroides and four strains of Clostridium was studied by use of resting cells and cell-free culture supernatants. Bacteroids strains yielded active cells but showed relatively low bile salt hydrolase (BSH) activity in the culture supernatants while the reverse was the case for the spore-forming clostridial strains. BSH was formed constitutively and was oxygen insensitive. The optimum pH was between 4.5 and 5.0. Marked substrate specificity was found in two strains, one Clostridium and one Bacteroides, which showed restricted activity against taurine conjugates. Bacteroides in general attacked the taurine conjugates of dihydroxy bile acids more readily than the trihydroxy taurine conjugates. Deconjugated bile acid moieties were further modified by some resting cells, depending on the bacterial strain while no enzymatic activity other than that of BSH was found in the culture supernatants. Cells of B. fragilis 2536 performed 7 alpha-dehydrogenation when the pH of the medium allowed the reaction, and this oxidative process was markedly enhanced in the presence of an abundant supply of oxygen as a terminal electron acceptor. C. perfringens PB 6K produced the 3- keto product in addition to the 3 beta-hydroxy derivative of the liberated bile acids and the formation of the latter derivative seemed to take place without preliminary deconjugation.     

Cloning and expression of cDNA encoding hamster liver 3-hydroxyhexobarbital/17β(3α)-hydroxysteroid dehydrogenase 1

Using RACE techniques we have cloned and sequenced one of the hamster liver 3-hydroxy-hexobarbital dehydrogenases which catalyze not only cyclic alcohols but also 17β-hydroxy-steroids and 3α-hydroxysteroids. The gene specific primers to 3-hydroxyhexobarbital dehydrogenase 1 (G2) were synthesized on the basis of its partial peptide sequences. The sequence of full length cDNA generated by 3′- and 5′-RACE PCR consisted of 1225 nucleotides including an open reading frame of 972 nucleotides encoding a protein of 323 amino acids. The deduced amino acid sequence matched exactly with the partial peptide sequences of hamster liver 3-hydroxyhexobarbital dehydrogenase 1 (G2). The sequence showed 84.5% identity to mouse liver 17β-dehydrogenase(A-specific), and 74–76% identity to human liver bile acid binding protein/3α-hydroxysteroid dehydrogenase (DD2), human liver 3α-hydroxysteroid dehydrogenase type I (DD4) and type II (DD3), and rabbit ovary 20α-hydroxysteroid dehydrogenase. The protein contains catalytic residues of aldo-keto reductases, Asp50, Tyr55, Lys84, His117. These results suggest that the hamster liver 3-hydroxyhexobarbital/17β(3α)-hydroxysteroid dehydrogenase belongs to aldo-keto reductase superfamily. The insert containing the full-length cDNA of 3-hydroxyhexobarbital dehydrogenase and vector specific overhang produced by PCR was annealed with pET-32 Xa/LIC vector. The plasmid was transformed into BL21 (DE3) cells containing pLysS. The recombinant enzyme was induced 1 mM IPTG. The expressed enzyme was produced as fusion protein and purified by nickel chelating affinity chromatography followed by POROS CM column chromatography and superdex 75 gel filtration. Molecular weight of the recombinant enzyme fused thioredoxin and his•tag was about 55 000 and that was 35 000 after Factor Xa protease treatment. The recombinant enzyme dehydrogenated 3-hydroxy-hexobarbital, 1-acenaphthenol, 2-cyclohexen-1-ol, testosterone, glycolithocholic acid as well as the native enzyme purified from hamster liver.     

One-step synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid using a multienzymatic system

12-ketoursodeoxycholic acid (12-keto-UDCA) is a key intermediate for the synthesis of ursodeoxycholic acid (UDCA), an important therapeutic agent for non-surgical treatment of human cholesterol gallstones and various liver diseases. The goal of this study is to develop a new enzymatic route for the synthesis 12-keto-UDCA based on a combination of NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH, EC 1.1.1.201) and NADH-dependent 3α-hydroxysteroid dehydrogenase (3α-HSDH, EC 1.1.1.50). In the presence of NADPH and NADH, the combination of these enzymes has the capacity to reduce the 3-carbonyl- and 7-carbonyl-groups of dehydrocholic acid (DHCA), forming 12-keto-UDCA in a single step. For cofactor regeneration, an engineered formate dehydrogenase, which is able to regenerate NADPH and NADH simultaneously, was used. All three enzymes were overexpressed in an engineered expression host Escherichia coli BL21(DE3)Δ7α-HSDH devoid of 7α-hydroxysteroid dehydrogenase, an enzyme indigenous to E. coli, in order to avoid formation of the undesired by-product 12-chenodeoxycholic acid in the reaction mixture. The stability of enzymes and reaction conditions such as pH value and substrate concentration were evaluated. No significant loss of activity was observed after 5 days under reaction condition. Under the optimal condition (10 mM of DHCA and pH 6), 99 % formation of 12-keto-UDCA with 91 % yield was observed.     

Convenient Non-Chromatographic Assays for the Microbial Deconjugation and 7α-OH Bioconversion of Taurocholate

We described two convenient assay methods to estimate bile acid deconjugation and bile acid bioconversion at the 7alpha-OH position by individual microorganisms grown in media containing taurocholic acid. The methods are based on (i) a selective chemical assay for taurine conjugates previously described and (ii) the use of a cell-free preparation of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli to directly quantify 7alpha-OH groups. These non-chromatographic approaches have been applied to the study of three model strains of intestinal organisms, E. coli, Bacteroides fragilis, and Clostridium perfringens, grown in standard media in the presence of purified tritiated taurocholate. Assay results were confirmed by thin-layer chromatography solvent systems designed to separate conjugated from unconjugated bile acid and unmodified cholic acid nucleus from 7alpha-OH bioconversion product(s) (primarily 3alpha, 12alpha dihydroxy, 7-keto-cholanoic acid). In addition, 7alpha-hydroxysteroid dehydrogenase activity was demonstrated in cell-free extracts of all three organisms. Of the three organisms, only C. perfringens was demonstrated to (i) deconjugate taurocholic acid, (ii) contain 3alpha-hydroxysteroid dehydrogenase activity, (iii) convert cholic acid into at least five labeled metabolites visible on thin-layer chromatography, and (iv) catalyze significant tritium exchange with water in the medium.     

Molecular cloning of rat liver 3α-hydroxysteroid dehydrogenase and identification of structurally related proteins from rat lung and kidney

3α-Hydroxysteroid dehydrogenase and related enzymes play important roles in the metabolism of endogenous compounds including androgens, corticosteroid, prostaglandins and bile acids, as well as drugs and xenobiotics such as benzo(a)pyrene. Complementary DNA clones encoding 3α-hydroxysteroid dehydrogenase were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. A full-length cDNA clone of 1286 base pairs contained an open reading frame encoding a protein of 322 amino acids with an estimated M(w) of 37 kD. When expressed in E. coli, the encoded protein migrated to the same position on SDS-polycrylamide gels as the enzyme in rat liver cytosols. The protein expressed in bacteria was highly active in androsterone oxidation in the presence of NAD as cofactor and this activity was inhibited by indomethacin, a potent inhibitor of 3α-hydroxysteroid dehydrogenase. The predicted amino acid sequence of 3α-hydroxysteroid d dehydrogenase was related to sequences of several other aldo-keto reductases such as bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase and frog lens epsilon-crystallin, suggesting that these proteins belong to the same gene family. Recently, we have found that monoclonal antibodies against 3α-hydroxysteroid dehydrogenase also recognized multiple antigenically related proteins in rat lung, kidney and testis. Further screening of liver, lung and kidney cDNA libraries using these monoclonal antibodies as probes resulted in the isolation of additional five different cDNAs encoding proteins with high degree of structural homology to rat liver 3α-hydroxysteroid dehydrogenase.     

In search of sustainable chemical processes: cloning, recombinant expression, and functional characterization of the 7α- and 7β-hydroxysteroid dehydrogenases from Clostridium absonum

Nicotinamide adenine dinucleotide phosphate-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenases (7β-HSDH) from Clostridium absonum catalyze the epimerization of primary bile acids through 7-keto bile acid intermediates and may be suitable as biocatalysts for the synthesis of bile acids derivatives of pharmacological interest. C. absonum 7α-HSDH has been purified to homogeneity and the N-terminal sequence has been determined by Edman sequencing. After PCR amplifications of a gene fragment with degenerate primers, cloning of the complete gene (786?nt) has been achieved by sequencing of C. absonum genomic DNA. The sequence coding for the 7β-HSDH (783?nt) has been obtained by sequencing of the genomic DNA region flanking the 5' termini of 7α-HSDH gene, the two genes being contiguous and presumably part of the same operon. After insertion in suitable expression vectors, both HSDHs have been successfully produced in recombinant form in Escherichia coli, purified by affinity chromatography and submitted to kinetic analysis for determination of Michaelis constants (K (m)) and specificity constants (k (cat)/K (m)) in the presence of various bile acids derivatives. Both enzymes showed a very strong substrate inhibition with all the tested substrates. The lowest K (S) values were observed with chenodeoxycholic acid and 12-ketochenodeoxycholic acid as substrates in the case of 7α-HSDH, whereas ursocholic acid was the most effective inhibitor of 7β-HSDH activity.     

Flow injection determination of glucose,bile acid and ATP using immobilized enzyme reactor and chemiluminescent assay of NAD(P)H

We have developed a chemiluminescent flow injection method for analysis of bile acid, glucose and ATP using the chemiluminescent assay of NADH using 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperoxidase (m-POD) system and immobilized enzyme reactors such as 3α-hydroxysteroid dehydrogenase, glucosedehydrogenase, hexokinase and glucose-6-phosphate dehydrogenase. The standard curves were obtained in the range of 5 ～ 100 pmol for bile acid, 0.5 ～ 5.0 nmol for glucose and 10^?7 ～ 10^?5 mol/L for ATP. The coefficient of variation for each assay was not more than 4.1% for bile acid, 2.3% for glucose and 5.3% for ATP, respectively.     

Bile acid biotransformation rates of selected gram-positive and gram-negative intestinal anaerobic bacteria.

Treatment of whole cell suspensions of $\text{Eubacterium aerofaciens}$ and $\text{Bacteroides fragilis}$ with lysozyme resulted in a marked increase (>100-fold) in the rates of biotransformation of cholate to 7-ketodeoxycholate (7-KD) in the former but only a 2-fold increase in the latter bacterium. In $\text{B. fragilis}$ the total activity of both NAD-dependent 7-α-hydroxysteroid dehydrogenase (7-α-OHSDH) and bile salt hydrolase (BSH) increase markedly during the stationary growth phase. Both enzymes were found in the spheroplast lysate and the Triton-soluble washed membrane fractions but only BSH was found in the spheroplast medium.     

Enzymatic reduction of dehydrocholic acid to 12-ketochenodeoxycholic acid with NADH regeneration

12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from dehydrocholic acid. The specific reduction of dehydrocholic with NADH was catalysed by 3α-hydroxysteroid dehydrogenase (3α-hydroxysteroid: NAD(P)⁺ oxidoreductase, EC 1.1.1.50) and 7α-hydroxysteroid dehydrogenase (7α-hydroxysteroid:NAD⁺ 7-oxidoreductase, EC 1.1.1.159). Cofactor regeneration was obtained through the formate dehydrogenase (formate:NAD⁺ oxidoreductase, EC 1.2.1.2) catalysed oxidation of formate. Complete transformation of dehydrocholic acid to the 12-keto derivative was achieved with a coenzyme turnover number up to 1200. No steroid by-products were detected by high performance liquid chromatography and thin layer chromatography. The process yielded 9 g product l^?1 in 66–84 h. The high purity of the enzymatically prepared 12-ketochenodeoxycholic acid should drastically reduce the formation of the toxic by-product lithocholic acid, which occurs in the synthesis of chenodeoxycholic acid when using chemical methods alone.     

Hepatic reduction of the secondary bile acid 7-oxolithocholic acid is mediated by 11β-hydroxysteroid dehydrogenase 1

The oxidized bile acid 7-oxoLCA (7-oxolithocholic acid), formed primarily by gut micro-organisms, is reduced in human liver to CDCA (chenodeoxycholic acid) and, to a lesser extent, UDCA (ursodeoxycholic acid). The enzyme(s) responsible remained unknown. Using human liver microsomes, we observed enhanced 7-oxoLCA reduction in the presence of detergent. The reaction was dependent on NADPH and stimulated by glucose 6-phosphate, suggesting localization of the enzyme in the ER (endoplasmic reticulum) and dependence on NADPH-generating H6PDH (hexose-6-phosphate dehydrogenase). Using recombinant human 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), we demonstrate efficient conversion of 7-oxoLCA into CDCA and, to a lesser extent, UDCA. Unlike the reversible metabolism of glucocorticoids, 11β-HSD1 mediated solely 7-oxo reduction of 7-oxoLCA and its taurine and glycine conjugates. Furthermore, we investigated the interference of bile acids with 11β-HSD1-dependent interconversion of glucocorticoids. 7-OxoLCA and its conjugates preferentially inhibited cortisone reduction, and CDCA and its conjugates inhibited cortisol oxidation. Three-dimensional modelling provided an explanation for the binding mode and selectivity of the bile acids studied. The results reveal that 11β-HSD1 is responsible for 7-oxoLCA reduction in humans, providing a further link between hepatic glucocorticoid activation and bile acid metabolism. These findings also suggest the need for animal and clinical studies to explore whether inhibition of 11β-HSD1 to reduce cortisol levels would also lead to an accumulation of 7-oxoLCA, thereby potentially affecting bile acid-mediated functions.     

Study of human placental estradiol-17β dehydrogenase/20α-hydroxysteroid dehydrogenase by preparative disc-gel electrophoresis

The soluble enzyme, estradiol-17β dehydrogenase from human term placenta, appears to co-purify with a second soluble enzyme, 20α-hydroxysteroid dehydrogenase. The enzyme, which had been partially purified by affinity chromatrography, fractionated on a preparative electrophoresis gel to a homogeneous preparation containing both estradiol-17β dehydrogenase and 20α-hydroxysteroid dehydrogenase activities in a ratio of ～100:1. Analytical polyacrylamide disc-gels resolved this homogeneous preparation as a single band by both protein and activity staining techniques. Homogeneous enzyme inactivated and affinity-radioalkylated by 16α-[2′-su14C]bromoacetoxyprogesterone or 16α-[2′-su14C] bromoacetoxyestradiol 3-methyl ether, and when analyzed by SDS disc-gel electrophoresis, gave a single protein band which corresponded identically to the radioactivity peaks. These observations support the hypothesis that estradiol-17β dehydrogenase and 20α-hydroxysteroid dehydrogenase represent dual oxidoreductase activity in one enzyme.Preparative disc-gel electrophoresis, a technique which has not been previously adapted to purification of these human placental enzyme activities, was useful to rapidly (3 days) effect a 15-fold enrichment of the estradiol-17β dehydrogenase specific activity from “heat-treated cytosol”. Thus, laboratory-scale preparative disc-gel electrophoresis is useful for rapid, small-scale enrichment of this soluble enzyme.     

Oxidation of omega-hydroxylated fatty acids and steroids by alcohol dehydrogenase

Recrystallized alcohol dehydrogenase from horse liver was found to oxidize 17-hydroxystearic acid into 17-oxostearic acid, the 17-L-enantiomer faster than the 17-D-enantiomer. Alone at high pH or in combination with aldehyde dehydrogenase, the alcohol dehydrogenase also catalyzed conversion of 18-hydroxystearic acid into 1, 18-octadecadioic acid and 5β-cholestane-3α,7α,12α,26-tetrol into 3α,7α,12α-trihydroxy-5β-cholestanoic acid. All the activities as well as the ethanol dehydrogenase activity disappeared after specific carboxymethylation of a single cystein residue at the active site of alcohol dehydrogenase. These results conclusively show that alcohol dehydrogenase itself has ω-hydroxyfatty acid dehydrogenase activity and ω-hydroxysteroid dehydrogenase activity.