How 7α- and 7β-hydroxysteroid dehydrogenases from Clostridium absonum enable sustainable production of vital liver disease treatments
Deep within our intestinal tract, trillions of microbial inhabitants perform silent chemical transformations that maintain our health in ways we're only beginning to understand.
Among these invisible workers are specialized enzymes that transform bile acids—crucial digestive compounds that also serve as potent medicines for liver disease. For decades, the primary treatment for primary biliary cholangitis, a chronic liver condition, has been ursodeoxycholic acid (UDCA), a bile acid traditionally obtained from bear bile or through complex chemical synthesis. Both methods present significant challenges, from ethical concerns to environmental pollution.
7α-HSDH and 7β-HSDH work in tandem to transform abundant bile acids into scarce therapeutic ones.
Offers a green alternative to traditional methods that rely on bear bile or chemical synthesis.
Bile acids are essential biological molecules our livers produce from cholesterol. They act as natural "detergents" that help emulsify and absorb dietary fats. Beyond this digestive function, certain bile acids have emerged as potent pharmaceutical agents.
Ursodeoxycholic acid (UDCA) and its conjugated form tauroursodeoxycholic acid (TUDCA) stand out for their remarkable therapeutic potential.
These bile acids do more than just treat liver disease—recent research has revealed they can prevent vision and hearing loss, slow retinal degeneration, and even show promise against neurodegenerative diseases like Alzheimer's and Parkinson's due to their ability to cross the blood-brain barrier .
Based on research findings 3
The magic of converting abundant primary bile acids into scarce therapeutic ones happens through a precise biochemical dance performed by two microbial enzymes:
This elegant transformation occurs through a 7-keto bile acid intermediate and represents one of nature's sophisticated solutions to chemical synthesis 1 . Both enzymes belong to the short-chain dehydrogenase/reductase (SDR) family and often share similar structural features, despite their different stereoselectivities 1 .
The journey to harness these enzymes begins with identifying their genetic blueprints. Researchers screen bacterial genomes, particularly from gut microorganisms known to modify bile acids. Clostridium absonum has emerged as a particularly rich source, with scientists successfully identifying the genes encoding both 7α- and 7β-HSDH from this bacterium .
Through genome mining approaches and tools like the EnzymeMiner platform, researchers can rapidly search growing genomic databases for promising enzyme candidates without laborious engineering 3 . This bioinformatics-driven approach has dramatically accelerated the discovery process.
Once identified, the target genes are inserted into specialized expression vectors—DNA molecules designed to transport and express foreign genes in host organisms. The pET series of plasmids, particularly pET-28a(+), has become a workhorse for this purpose, containing specific tags that simplify subsequent protein purification 3 .
These engineered DNA constructs are then introduced into bacterial host systems, with Escherichia coli BL21(DE3) being the preferred choice due to its well-characterized genetics, rapid growth, and ability to efficiently produce foreign proteins 2 3 .
The real magic happens when these engineered bacteria are induced to express the cloned genes, leading to massive production of the target enzymes. Scientists then break open the bacterial cells and purify the enzymes using affinity tags engineered into the original DNA construct.
Common purification systems include the GST gene fusion system, which allows for mild purification conditions without denaturing the delicate enzyme structures . After purification, researchers obtain the pristine enzymes necessary for detailed characterization and application.
Once purified, the enzymes undergo rigorous characterization to establish their biochemical "personalities." For the 7α- and 7β-HSDHs from Clostridium absonum, we know:
Based on characterization data
For industrial applications, enzymes must be not only active but also stable under process conditions. The 7α-HSDH from Shewanella morhuae exemplifies the robust characteristics researchers seek—it maintains exceptional thermal stability with a half-life of 120 hours at 35°C 3 .
Enzyme activity is strongly influenced by environmental conditions. Through systematic testing, researchers establish optimal pH ranges and temperature profiles for maximum activity. The 7β-HSDH from Ruminococcus gnavus, for instance, shows a strong preference for the UDCA-producing reaction, with its forward reaction rate approximately 55-fold higher than the reverse 4 .
Kinetic analysis reveals how efficiently enzymes perform their catalytic tasks. By measuring parameters like Km (indicating substrate affinity) and kcat (catalytic turnover), researchers can quantify enzyme performance and identify the most promising candidates.
| Enzyme Source | Substrate | Km (mM) | kcat (s⁻¹) | kcat/Km (mM⁻¹s⁻¹) | Citation |
|---|---|---|---|---|---|
| 7β-HSDH from R. gnavus | 7-oxo-LCA | Not specified | Not specified | Forward reaction favored 55:1 | 4 |
| 7α-HSDH from C. absonum | TCDCA | 0.24 | Not specified | Not specified | |
| 7α-HSDH from C. absonum (+ bilirubin) | TCDCA | 0.63 | Not specified | Not specified | |
| 7β-HSDH from C. absonum | TUDCA | 1.14 | Not specified | Not specified | |
| 7β-HSDH from C. absonum (+ bilirubin) | TUDCA | 1.87 | Not specified | Not specified |
The kinetic data demonstrates how bilirubin inhibits both enzymes by reducing their affinity for substrates, as evidenced by the increased Km values . Understanding these parameters is essential for developing effective industrial processes.
While exploring sustainable UDCA production from waste chicken bile—a rich source of the substrate taurochenodeoxycholic acid (TCDCA)—researchers noticed a puzzling phenomenon: the catalytic efficiency of their enzyme system dropped precipitously. Suspecting that bilirubin, a yellow pigment abundant in chicken bile, might be inhibiting the enzymes, they designed a comprehensive experiment to investigate this effect .
The researchers hypothesized that bilirubin was directly interfering with enzyme function, possibly by binding to the enzymes and altering their structure or blocking substrate access. To test this, they purified both 7α- and 7β-HSDHs and exposed them to varying concentrations of bilirubin (0-1 mM) while measuring changes in enzyme activity, kinetics, and structure .
Based on experimental data
The investigation employed a sophisticated combination of techniques:
The findings revealed a dramatic, dose-dependent inhibition by bilirubin. At 1 mM bilirubin concentration, 7α-HSDH retained less than 40% of its original activity, while 7β-HSDH was even more severely affected, retaining a mere 18% of its activity .
| Parameter | 7α-HSDH | 7β-HSDH |
|---|---|---|
| Relative Activity with 1 mM Bilirubin | <40% | 18% |
| Km without Bilirubin | 0.24 mM (TCDCA) | 1.14 mM (TUDCA) |
| Km with Bilirubin | 0.63 mM (TCDCA) | 1.87 mM (TUDCA) |
| Structural Changes | Altered secondary structure | Altered secondary structure |
| Binding Confirmation | UV-Vis, Fluorescence, CD | UV-Vis, Fluorescence, CD |
This research transcended mere observation of inhibition—it provided a mechanistic understanding of the phenomenon. By identifying bilirubin as a potent inhibitor and characterizing its mode of action, the study offered practical guidance for process optimization: bilirubin removal is essential for efficient biotransformation of chicken bile.
The study and application of 7α- and 7β-HSDHs relies on a collection of specialized reagents and materials that enable every stage of research, from gene discovery to industrial application.
| Reagent/Material | Function and Significance | Examples/Specifications |
|---|---|---|
| Expression Vectors | DNA vehicles for gene cloning and expression | pET-28a(+), GST fusion vectors |
| Host Organisms | Living systems for enzyme production | E. coli BL21(DE3), E. coli JM109 |
| Substrates | Molecules transformed by enzymes | CDCA, 7-oxo-LCA, TCDCA |
| Cofactors | Essential helper molecules for catalysis | NAD+, NADP+, NADPH |
| Purification Systems | Tools for enzyme isolation and cleaning | GST gene fusion system, His-tag systems |
| Chromatography Materials | Separation and analysis tools | HPLC systems, GC-MS, TLC plates |
| Culture Media | Nutrient sources for microbial growth | GAM broth, LB medium, M9 medium |
This toolkit continues to evolve with advancing technology. For instance, the development of EnzymeMiner and other bioinformatics platforms has revolutionized the discovery phase, allowing researchers to identify promising enzyme candidates from genomic databases without laborious laboratory screening 3 . Similarly, high-throughput screening methods have accelerated the engineering of improved enzyme variants.
The journey of 7α- and 7β-hydroxysteroid dehydrogenases from obscure bacterial enzymes to green pharmaceutical tools exemplifies how understanding nature's molecular machinery can address practical human needs.
Through cloning, recombinant expression, and detailed characterization, scientists have transformed these microbial proteins into efficient biocatalysts for sustainable medicine production.
Enabling efficient UDCA production without bear bile or polluting chemical processes represents a triumph of sustainable pharmaceutical manufacturing.
Recent breakthroughs demonstrate remarkable efficiency with conversion of 200 mM CDCA to 7-oxo-LCA with a 78% isolated yield and impressive space-time yield of 1418 g L⁻¹ d⁻¹ 3 .
Potential applications are expanding as new therapeutic benefits of UDCA and TUDCA continue to emerge, particularly in neurodegenerative disease treatment .
As we look ahead, the integration of artificial intelligence for enzyme design, along with continued exploration of natural diversity through metagenomic mining, promises to unlock even more powerful biocatalysts.
The story of these remarkable enzymes reminds us that sometimes the most sophisticated solutions to human challenges can be found in nature's smallest creations—we need only learn how to harness them.
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