7β-hydroxysteroid Dehydrogenase Research Articles

Personalized Microbial Fingerprint Associated with Differential Glycemic Effects of a Whole Grain Rye Intervention on Chinese Adults.

This study aims to identify the gut enterotypes that explain differential responses to intervention with whole grain rye by proposing an "enterotype - metabolic" model. A 12-week randomized controlled trial is conducted in Chinese adults, with 79 subjects consuming whole grain products with fermented rye bran (FRB) and 77 consuming refined wheat products in this exploratory post-hoc analysis. Responders or non-responders are identified according to whether blood glucose decreased by more than 10% after rye intervention. Compared to non-responders, responders in FRB have higher baseline Bacteroides (p < 0.001), associated with reduced blood glucose (p < 0.001), increased Faecalibacterium (p = 0.020) and Erysipelotrichaceae_UCG.003 (p = 0.022), as well as deceased 7β-hydroxysteroid dehydrogenase (p = 0.033) after intervention. The differentiated gut microbiota and metabolites between responders and non-responders after intervention are enriched in aminoacyl-tRNA biosynthesis. The work confirms the previously suggested importance of microbial enterotypes in differential responses to whole grain interventions and supports taking enterotypes into consideration for improved efficacy of whole grain intervention for preventing type 2 diabetes. Altered short-chain fatty acids and bile acid metabolism might be a potential mediator for the beneficial effects of whole grain rye on glucose metabolism.

Boosting the Biosynthesis of Ursodeoxycholic Acid via a Novel 7β-Hydroxysteroid Dehydrogenase from Olsenella sp. and Biotransformation Systematic Optimization

Boosting the Biosynthesis of Ursodeoxycholic Acid via a Novel 7β-Hydroxysteroid Dehydrogenase from <i>Olsenella</i> sp. and Biotransformation Systematic Optimization

Machine-Learning-Guided Engineering of an NADH-Dependent 7β-Hydroxysteroid Dehydrogenase for Economic Synthesis of Ursodeoxycholic Acid.

Enzymatic synthesis of ursodeoxycholic acid (UDCA) catalyzed by an NADH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH) is more economic compared with an NADPH-dependent 7β-HSDH when considering the much higher cost of NADP+/NADPH than that of NAD+/NADH. However, the poor catalytic performance of NADH-dependent 7β-HSDH significantly limits its practical applications. Herein, machine-learning-guided protein engineering was performed on an NADH-dependent Rt7β-HSDHM0 from Ruminococcus torques. We combined random forest, Gaussian Naïve Bayes classifier, and Gaussian process regression with limited experimental data, resulting in the best variant Rt7β-HSDHM3 (R40I/R41K/F94Y/S196A/Y253F) with improvements in specific activity and half-life (40 °C) by 4.1-fold and 8.3-fold, respectively. The preparative biotransformation using a "two stage in one pot" sequential process coupled with Rt7β-HSDHM3 exhibited a space-time yield (STY) of 192 g L-1 d-1, which is so far the highest productivity for the biosynthesis of UDCA from chenodeoxycholic acid (CDCA) with NAD+ as a cofactor. More importantly, the cost of raw materials for the enzymatic production of UDCA employing Rt7β-HSDHM3 decreased by 22% in contrast to that of Rt7β-HSDHM0, indicating the tremendous potential of the variant Rt7β-HSDHM3 for more efficient and economic production of UDCA.

Analysis of gut microbiome, host genetics, and plasma metabolites reveals gut microbiome-host interactions in the Japanese population

Interaction between the gut microbiome and host plays a key role in human health. Here, we perform a metagenome shotgun-sequencing-based analysis of Japanese participants to reveal associations between the gut microbiome, host genetics, and plasma metabolome. A genome-wide association study (GWAS) for microbial species (n= 524) identifies associations between the PDE1C gene locus and Bacteroides intestinalis and between TGIF2 and TGIF2-RAB5IF gene loci and Bacteroides acidifiaciens. In a microbial gene ortholog GWAS, agaE and agaS, which are related to the metabolism of carbohydrates forming the blood group A antigen, are associated with blood group A in a manner depending on the secretor status determined by the East Asian-specific FUT2 variant. A microbiome-metabolome association analysis (n= 261) identifies associations between bile acids and microbial features such as bile acid metabolism gene orthologs including bai and 7β-hydroxysteroid dehydrogenase. Our publicly available data will be a useful resource for understanding gut microbiome-host interactions in an underrepresented population.

The Impact of Bilirubin on 7α- and 7β-Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

7α- and 7β-hydroxysteroid dehydrogenases (HSDHs) are enzymes that can catalyze the isomerization of hydroxyl groups at site seven of bile acids. In a previous study, we found that the activities of 7α- and 7β-HSDHs can be inhibited by bilirubin. In order to clarify the impact, the effects of bilirubin on enzymes were studied by kinetics, spectrum, and docking analysis. The relative activity of 7α-HSDH remained less than 40% under 1 mM bilirubin, and only 18% activity of 7β-HSDH kept in the same condition. Using taurochenodeoxycholic acid (TCDCA) as substrate, the Km of 7α-HSDH was up to 0.63 mM from 0.24 mM after binding with bilirubin and the Km of 7β-HSDH rose from 1.14 mM to 1.87 mM for the catalysis of tauroursodeoxycholic acid (TUDCA). The affinity of 7α- and 7β-HSDHs to substrates decreased with the effect of bilirubin. The binding of bilirubin with 7α- or 7β-HSDHs was analyzed by UV–vis, fluorescence, and circular dichroism (CD) spectroscopy. The results reflected that bilirubin caused a slight change in the secondary structure of 7α- or 7β-HSDHs, and the changes were correlated with the ratio of bilirubin to enzymes. Ten candidate molecular docking results were presented to reflect the binding of bilirubin with 7α- or 7β-HSDHs and to explore the inhibition mechanism. This research provides a more in-depth understanding of the effect of bilirubin on 7α- and 7β-HSDHs.

Clean enzymatic production of ursodeoxycholic acid enabled by a newly identified NADH-dependent 7β-hydroxysteroid dehydrogenase

7β-Hydroxysteroid dehydrogenases (7β-HSDHs) play an important role in the enzymatic synthesis of ursodeoxycholic acid (UDCA), which is a value-added compound with a range of pharmacological activities. Since the cofactor NADH is much cheaper than NADPH, the production of UDCA using an NADH-dependent 7β-HSDH has better prospects than that using an NADPH-dependent 7β-HSDH. However, a major bottleneck in UDCA biosynthesis is the poor catalytic activity of the current NADH-dependent 7β-HSDHs. In this work, a new and natively NADH-dependent Rs7β-HSDH from Roseococcus sp. was identified, which showed a high specific activity of 63.3 U mg−1protein toward 7-oxo-lithocholic acid, with a catalytic efficiency (kcat/KM) of 515 mM–1 s–1. In a preparative biotransformation (100-mL scale) using Rs7β-HSDH and an NAD+-dependent 7α-HSDH, with O2/NOX and HCOO−/FDH systems for the regeneration of cofactors (NAD+ and NADH), 25 mM chenodeoxycholic acid was completely converted to UDCA in one-pot two-step cascade, with an 80% isolated yield and a total turnover number (TTN) of 6586 for Rs7β-HSDH. The environmental factor (E-factor) of this process was 8.37 when water was excluded, much lower than those obtained from other processes reported so far, indicating a great potential of this Rs7β-HSDH for more sustainable and cleaner enzymatic production of UDCA.

Sequence and structure-guided discovery of a novel NADH-dependent 7β-hydroxysteroid dehydrogenase for efficient biosynthesis of ursodeoxycholic acid

7β-Hydroxysteroid dehydrogenases (7β-HSDHs) have attracted increasing attention due to their crucial roles in the biosynthesis of ursodeoxycholic acid (UDCA). However, most published 7β-HSDHs are strictly NADPH-dependent oxidoreductases with poor activity and low productivity. Compared with NADPH, NADH is more stable and cheaper, making it the more popular cofactor for industrial applications of dehydrogenases. Herein, by using a sequence and structure-guided genome mining approach based on the structural information of conserved cofactor-binding motifs, we uncovered a novel NADH-dependent 7β-HSDH (Cle7β-HSDH). The Cle7β-HSDH was overexpressed, purified, and characterized. It exhibited high specific activity (9.6 U/mg), good pH stability and thermostability, significant methanol tolerance, and showed excellent catalytic efficiencies (kcat/Km) towards 7-oxo-lithocholic acid (7-oxo-LCA) and NADH (70.8 mM-1s−1 and 31.8 mM-1s−1, respectively). Molecular docking and mutational analyses revealed that Asp42 could play a considerable role in NADH binding and recognition. Coupling with a glucose dehydrogenase for NADH regeneration, up to 20 mM 7-oxo-LCA could be completely transformed to UDCA within 90 min by Cle7β-HSDH. This study provides an efficient approach for mining promising enzymes from genomic databases for cost-effective biotechnological applications.

An Overview of 7α- and 7β-Hydroxysteroid Dehydrogenases: Structure, Specificity and Practical Application.

7α-Hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase are key enzymes involved in bile acid metabolism. They catalyze the epimerization of a hydroxyl group through 7-keto bile acid intermediates. Basic research of the two enzymes has focused on exploring new enzymes and the structure-function relationship. The application research focused on the in vitro biosynthesis of bile acid drugs and the exploration and improvement of their catalytic ability based on molecular engineering. This article summarized the primary and advanced structural characteristics, specificities, biochemical properties, and applications of the two enzymes. The emphasis is also given to obtaining novel 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase that are thermally stable and active in the presence of organic solvents, high substrate concentration, and extreme pH values. To achieve these goals, enzyme redesigning based on protein engineering and genomics may be the most useful approaches.

Molecular Mechanism Study on Stereo-Selectivity of α or β Hydroxysteroid Dehydrogenases

Hydroxysteroid dehydrogenases (HSDHs) are from two superfamilies of short-chain dehydrogenase (SDR) and aldo–keto reductase (AKR). The HSDHs were summarized and classified according to their structural and functional differences. A typical pair of enzymes, 7α–hydroxysteroid dehydrogenase (7α–HSDH) and 7β–hydroxysteroid dehydrogenase (7β–HSDH), have been reported before. Molecular docking of 7-keto–lithocholic acid(7–KLA) to the binary of 7β–HSDH and nicotinamide adenine dinucleotide phosphate (NADP+) was realized via YASARA, and a possible binding model of 7β–HSDH and 7–KLA was obtained. The α side of 7–KLA towards NADP+ in 7β–HSDH, while the β side of 7–KLA towards nicotinamide adenine dinucleotide (NAD+) in 7α–HSDH, made the orientations of C7–OH different in products. The interaction between Ser193 and pyrophosphate of NAD(P)+ [Ser193–OG⋯3.11Å⋯O1N–PN] caused the upturning of PN–phosphate group, which formed a barrier with the side chain of His95 to make 7–KLA only able to bind to 7β–HSDH with α side towards nicotinamide of NADP+. A possible interaction of Tyr253 and C24 of 7–KLA may contribute to the formation of substrate binding orientation in 7β–HSDH. The results of sequence alignment showed the conservation of His95, Ser193, and Tyr253 in 7β–HSDHs, exhibiting a significant difference to 7α–HSDHs. The molecular docking of other two enzymes, 17β–HSDH from the SDR superfamily and 3(17)α–HSDH from the AKR superfamily, has furtherly verified that the stereospecificity of HSDHs was related to the substrate binding orientation.

Large-scale production of ursodeoxycholic acid from chenodeoxycholic acid by engineering 7α- and 7β-hydroxysteroid dehydrogenase.

7α- and 7β-hydroxysteroid dehydrogenases (HSDHs) are key biocatalysts for the biotransformation of ursodeoxycholic acid (UDCA) from chenodeoxycholic acid (CDCA). Various researches focused on heterogeneously expressed engineering enzymes to epimerize CDCA for UDCA, however not yet applied to further industrial application. In this work, we present the large-scale production of UDCA from CDCA by 7α- and 7β-HSDH enzymatic synthesis. Engineering enzymes were both successfully heterologous overexpressed in Escherichia coli BL21, and the effect of the enzymatic synthesis was investigated. The mass analysis (MS), IR spectrum, 1H NMR and 13C NMR were used to characterize the product. 500-L fermentor fermentation strategy producing a stable supply of HSDH and large-scale production process of UDCA in dozens kilogram class enabled industrial application.

Switching Cofactor Dependence of 7β-Hydroxysteroid Dehydrogenase for Cost-Effective Production of Ursodeoxycholic Acid

Dehydrogenases are widely employed as biocatalysts for the production of optically pure chemicals under mild conditions. Most dehydrogenases are nicotinamide cofactor (NADPH or NADH)-dependent oxid...

Continuous Production of Ursodeoxycholic Acid by Using Two Cascade Reactors with Co-immobilized Enzymes.

Ursodeoxycholic acid (UDCA) is an effective drug for the treatment of hepatitis. In this study, 7α-hydroxysteroid dehydrogenase (7α-HSDH) and lactate dehydrogenase (LDH), as well as 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH), were co-immobilized onto an epoxy-functionalized resin (ES-103) to catalyze the synthesis of UDCA from chenodeoxycholic acid (CDCA). Through optimizing the immobilization pH, time, and loading ratio of enzymes to resin, the specific activities of immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 were 43.2 and 25.8 U g-1 , respectively, which were 12- and 516-fold higher than that under the initial immobilization conditions. Continuous production of UDCA from CDCA was subsequently achieved by using immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 in two serial packed-bed reactors. The yield of UDCA reached nearly 100 % and lasted for at least 12 h in the packed-bed reactors, which was superior to that of the batchwise reaction. This efficient continuous approach developed herein might provide a feasible route for large-scale biotransformation of CDCA into UDCA.

Rapidly directional biotransformation of tauroursodeoxycholic acid through engineered Escherichia coli.

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±0.13g/L of TUDCA from 3.23g/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.

Structure of NADP+-bound 7β-hydroxysteroid dehydrogenase reveals two cofactor-binding modes.

In mammals, bile acids/salts and their glycine and taurine conjugates are effectively recycled through enterohepatic circulation. 7β-Hydroxysteroid dehydrogenases (7β-HSDHs; EC 1.1.1.201), including that from the intestinal microbe Collinsella aerofaciens, catalyse the NADPH-dependent reversible oxidation of secondary bile-acid products to avoid potential toxicity. Here, the first structure of NADP+ bound to dimeric 7β-HSDH is presented. In one active site, NADP+ adopts a conventional binding mode similar to that displayed in related enzyme structures. However, in the other active site a unique binding mode is observed in which the orientation of the nicotinamide is different. Since 7β-HSDH has become an attractive target owing to the wide and important pharmaceutical use of its product ursodeoxycholic acid, this work provides a more detailed template to support rational protein engineering to improve the enzymatic activities of this useful biocatalyst, further improving the yield of ursodeoxycholic acid and its other applications.

Discovery of tauroursodeoxycholic acid biotransformation enzymes from the gut microbiome of black bears using metagenomics

Tauroursodeoxycholic acid (TUDCA) has been used to treat many diseases effectively. 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) are two key enzymes that drive the efficient biosynthesis of TUDCA from taurochenodeoxycholic acid (TCDCA) in vitro. In this study, a metagenomic approach was used to isolate 7α- and 7β-HSDHs from fecal samples of black bears. Five new 7α-HSDHs and one new 7β-HSDH enzyme were discovered and identified from the gut microbiota of black bears, and four of them presented good enzymatic properties. Our data also suggest cooperation in the biotransformation of TUDCA by the gut microbiota in black bears. In conclusion, this work expands the natural enzyme bank of HSDHs, provides promising candidate enzymes for application in the biosynthesis TUDCA and the epimerization reaction of bile acids at the C-7 position, and provides a data set for the discovery of novel enzymes in the gut micriobiome of black bears.

Engineering 7β-Hydroxysteroid Dehydrogenase for Enhanced Ursodeoxycholic Acid Production by Multiobjective Directed Evolution.

Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile powder with multiple pharmacological functions. 7β-Hydroxysteroid dehydrogenase (HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7β-HSDHs reported commonly suffer from poor activity and thermostability, resulting in limited productivity of UDCA. In this study, a multiobjective directed evolution (MODE) strategy was proposed and applied to improve the activity, thermostability, and pH optimum of a 7β-HSDH. The best variant (V3-1) showed a specific activity 5.5-fold higher than and a half-life 3-fold longer than those of the wild type. In addition, the pH optimum of the variant was shifted to a weakly alkaline value. In the cascade reaction, the productivity of UDCA with V3-1 increased to 942 g L-1 day-1, in contrast to 141 g L-1 day-1 with the wild type. Therefore, this study provides a useful strategy for improving the catalytic efficiency of a key enzyme that significantly facilitated the bioproduction of UDCA.

Structural and biochemical insights into 7β-hydroxysteroid dehydrogenase stereoselectivity.

Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo- and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active-site accessibility, the bases of the specificity for NADP(+) , and the general architecture of the steroid binding site. Comparison with 7α-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the β-selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859-865. © 2016 Wiley Periodicals, Inc.

Preparing tauroursodeoxycholic acid (TUDCA) using a double-enzyme-coupled system

Tauroursodeoxycholic acid (TUDCA) has been used to treat many diseases effectively. TUDCA generation in vivo is a complex process involving five steps catalyzed by five enzymes in the enterohepatic circulation of bile salt. In this paper, we report a novel in vitro TUDCA preparation technology in which the epimerization of taurochenodeoxycholic acid (TCDCA) to TUDCA is directly catalyzed by immobilized 7α- and 7β-hydroxysteroid dehydrogenases (7α- and 7β-HSDH) in a double-enzyme-coupled system. One pair of oxidoreductases from Clostridium absonum DSM599, which was made up of 7α- and 7β-HSDH, was immobilized on a modified chitosan microsphere by separate immobilization and co-immobilization. The protein loading yields of 7α- and 7β-HSDH were 79.06% and 87.18%, and their activity yields were 57.23% and 53.97%, respectively. In the batch-wise reactions catalyzed by double-enzyme-coupled system, 72.76% of the TCDCA was transformed, and only 22.08% TUDCA was obtained on the basis of separate immobilization. The TUDCA yield was 62.49% under the catalytic effects of co-immobilized 7α- and 7β-HSDH microspheres, while it was only 41.23% based on the mixture of separate immobilized 7α- and 7β-HSDH microspheres. The enzyme catalytic technology described in this paper offers a potential application for synthesizing TUDCA and other high-value bile acid derivatives in vitro.

Dynamic mechanistic modeling of the multienzymatic one-pot reduction of dehydrocholic acid to 12-keto ursodeoxycholic acid with competing substrates and cofactors.

Ursodeoxycholic acid (UDCA) is a bile acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two-step reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid (12-keto-UDCA), which can be conducted in a multienzymatic one-pot process using 3α-hydroxysteroid dehydrogenase (3α-HSDH), 7β-hydroxysteroid dehydrogenase (7β-HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one-pot reduction which involves three enzymes, four different bile acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi-bi mechanism with EBQ-type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch-process was established by coupling rate equations and mass balances. Validation experiments of the one-pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions.

Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques

7β-Hydroxysteroid dehydrogenase (7β-HSDH) is a key enzyme for the efficient biosynthesis of ursodeoxycholic acid (UDCA), an effective pharmaceutical for primary biliary cirrhosis and human cholesterol gallstones. In this work, a new 7β-HSDH from Ruminococcus torques ATCC 35915, designated as 7β-HSDHRt, was identified and heterologously overexpressed in Escherichia coli for the enzymatic synthesis of ursodeoxycholic acid from chenodeoxycholic acid (CDCA). 7β-HSDHRt was firstly employed in one-pot mode together with 7α-HSDHCa, another NADPH-dependent 7α-HSDH from Clostridium absonum, to convert CDCA into UDCA without additional coenzyme regeneration. However, the final yield was limited to merely 73%, probably due to chemical equilibrium. Therefore, to enhance the UDCA yield, we alternatively adopted a two-step reaction strategy where the enzymes involved in the first reaction were simply heat-inactivated between the 1st-step reaction (dehydrogenation) and the 2nd-step reaction (hydrogenation), in order to prevent the undesired bioreduction of 7-oxo-LCA into CDCA in the 2nd step. Consequently, the analytic yield of UDCA was significantly improved up to above 98% at a substrate load of 10mM (ca. 4gL−1), without any detectable intermediate (7-oxo-LCA) as observed in the case of one-pot reaction.