Recombinant Rabbit Prostaglandin-E (2) 9-reductase

What is Rabbit Prostaglandin-E2 9-reductase and what is its physiological significance?

Prostaglandin-E2 9-reductase (PGE2 9-reductase) is a cytosolic, NADPH-dependent enzyme belonging to the aldo-keto reductase superfamily. In rabbit tissues, this enzyme has a molecular mass of approximately 36 kDa in the corpus luteum, though its mass varies in other tissues. The enzyme's primary function is catalyzing the reduction of the 9-keto group of PGE2 to form PGF2α, serving as a critical regulator of prostaglandin metabolism .

The physiological significance of this enzyme lies in its role in reproductive function. PGE2 9-reductase exhibits dual catalytic functionality, acting both as a PGE2 9-reductase and as a 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). This dual activity creates a direct biochemical link between prostaglandin and steroid metabolism . The enzyme appears to be a key factor in the cascade of events leading to corpus luteum regression in rabbits, particularly through controlling the balance between luteotropic PGE2 and luteolytic PGF2α .

How does PGE2 9-reductase differ between rabbit tissues?

PGE2 9-reductase demonstrates significant tissue specificity in rabbits, with distinct biochemical properties observed between different tissues. This tissue specificity suggests specialized adaptations for local functional requirements.

The renal enzyme is localized specifically in the renal cortex and demonstrates a broad pH profile with an optimum at pH 7.5. Kinetic studies of the kidney enzyme indicated a Km of 3.2 × 10⁻⁴ M for PGE2 . These differences in molecular weight and kinetic parameters between the corpus luteum and kidney enzymes strongly suggest that these are either different isoforms or the same protein with tissue-specific post-translational modifications. The values also differ from those obtained with similar enzymes from monkey brain tissue, further indicating the tissue-specific nature of PGE2 9-reductase across different sources .

What temporal patterns of PGE2 9-reductase activity occur during pregnancy and pseudopregnancy?

The activity of PGE2 9-reductase in rabbit corpus luteum follows distinct temporal patterns that differ between pregnancy and pseudopregnancy, suggesting a regulatory role in reproductive physiology:

In pseudopregnant rabbits:

• PGE2 9-reductase activity is significantly elevated on Days 10, 12, and 15 post-ovulation compared to Day 8

• This increased activity correlates with the period preceding corpus luteum regression, which occurs around Day 12 after hCG administration

In pregnant rabbits:

• PGE2 9-reductase activity increases only on Day 12

• Activity subsequently declines to basal levels on Days 13 and 15

• A significant difference in enzyme activity is observed between pregnant and pseudopregnant animals on Day 15

These temporal changes in enzyme activity correlate with PGF2α concentrations, which parallel PGE2 9-reductase activity. The distinct patterns between pregnancy and pseudopregnancy suggest that the regulation of PGE2 9-reductase activity may be a key mechanism controlling corpus luteum lifespan and function in rabbits. This enzyme likely participates in regulating the PGF2α/PGE2 ratio, which is critical for determining whether the corpus luteum will persist (during pregnancy) or regress (during pseudopregnancy) .

What are the optimal strategies for cloning and expressing rabbit PGE2 9-reductase in E. coli?

When designing expression systems for recombinant rabbit PGE2 9-reductase, researchers should implement a systematic approach that addresses both gene optimization and expression conditions:

Gene and Vector Design:

• Use codon optimization for E. coli expression, particularly considering that enzymes from eukaryotic sources often contain codons rarely used in E. coli

• Select appropriate expression vectors (pET series vectors are commonly recommended for enzymes)

• Include a removable affinity tag (His6 or GST) to facilitate purification

• Consider fusion with solubility-enhancing partners (MBP, SUMO, or Trx) if initial expression yields inclusion bodies

Expression Host Selection:

• BL21(DE) or Rosetta strains are preferred for enzyme expression

• If inclusion bodies persist, consider specialized strains like Origami or SHuffle that enhance disulfide bond formation

• For co-expression with chaperones, BL21(DE3) containing chaperone plasmids (e.g., pG-KJE8) can be employed

Expression Conditions Optimization:

• Perform small-scale expression trials varying temperature (16-37°C), IPTG concentration (0.1-1 mM), and induction time (4-20 hours)

• Lower temperatures (16-20°C) generally favor proper folding of aldo-keto reductases

• Include cofactors (NADPH) in growth media to potentially enhance proper folding

• Consider auto-induction media as an alternative to IPTG induction

The application of bioinformatics and modeling tools, used in only 19% of difficult-to-express enzyme studies, can significantly improve expression outcomes . Sequence alignment with successfully expressed homologs can identify potential problematic regions, while structural prediction tools can guide rational modifications to enhance solubility without compromising activity.

What purification methods are most effective for recombinant rabbit PGE2 9-reductase?

Based on successful purification strategies for native rabbit PGE2 9-reductase, the following multi-step protocol is recommended for the recombinant enzyme:

Step 1: Initial Capture

• If His-tagged: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• If tag-free: Red Sepharose CL-6B affinity chromatography, which proved highly effective for native enzyme purification

• For Red Sepharose purification, implementation of a sequential elution strategy is critical:

  • First elute with 1 mM NADH to remove non-specifically bound proteins

  • Then elute the active enzyme with 1 mM NADPH

  • This approach significantly improved purification efficiency for the native enzyme

Step 2: Intermediate Purification

• Ion exchange chromatography based on the enzyme's predicted isoelectric point

• Anion exchange (e.g., Q Sepharose) at pH 8.0 if the isoelectric point is below 7

• Similar approach was effectively used for bovine placental PGE2 9-reductase

Step 3: Polishing

• Size exclusion chromatography using Superdex 75 or equivalent

• This step ensures removal of aggregates and separation from proteins of different molecular weights

• Can also be used for buffer exchange into final storage buffer

Buffer Considerations:

• Include 1-5 mM DTT or β-mercaptoethanol throughout purification

• Add 10-20% glycerol to stabilize protein structure

• Maintain pH between 7.0-8.0 based on stability data from native enzyme

• Include 0.1-0.2 mM NADPH in storage buffer to maintain enzyme stability

This purification strategy can yield enzyme with >95% purity suitable for biochemical characterization and crystallization trials. When applied to the native corpus luteum enzyme, similar approaches achieved a 266-fold enrichment , while a 135-fold purification was reported for bovine placental enzyme .

How can solubility and proper folding be enhanced during recombinant expression?

Recombinant expression of enzymes like rabbit PGE2 9-reductase in E. coli frequently results in inclusion body formation. Several strategies can enhance solubility and proper folding:

Expression Condition Optimization:

• Reduce expression temperature to 16-20°C after induction

• Lower inducer concentration (0.1-0.2 mM IPTG)

• Extend expression time (16-24 hours) at reduced temperature

• Include osmolytes (5-10% glycerol, 0.5-1 M sorbitol) in culture medium

Genetic Modifications:

• Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

• Fusion with solubility-enhancing partners (MBP, SUMO, Trx)

• Remove hydrophobic regions or cysteine residues prone to misfolding

• Create truncated constructs guided by bioinformatic domain prediction

Lysis and Extraction Optimization:

• Use mild non-ionic detergents (0.1% Triton X-100) in lysis buffer

• Include stabilizing agents (5-10% glycerol, 0.5 M arginine)

• Add cofactors (NADPH) to promote proper folding during extraction

• Consider enzymatic lysis (lysozyme) rather than sonication to minimize aggregation

Refolding Strategies (if inclusion bodies persist):

• Solubilize inclusion bodies in 6 M guanidine hydrochloride

• Employ step-wise dialysis with decreasing denaturant concentration

• Include redox pairs (reduced/oxidized glutathione) to facilitate proper disulfide formation

• Add molecular chaperones during refolding

A systems biology approach integrating bioinformatics and experimental data can significantly improve outcomes for difficult-to-express enzymes like PGE2 9-reductase . Predictive tools can identify problematic regions, while high-throughput screening of expression conditions can accelerate optimization. The relative scarcity of bioinformatics application in recombinant enzyme expression (19% of studies) suggests substantial opportunities for improvement using these advanced approaches .

What are the optimal assay conditions for measuring recombinant PGE2 9-reductase activity?

Accurate measurement of recombinant rabbit PGE2 9-reductase activity requires carefully optimized assay conditions. Based on the biochemical properties reported for the native enzyme, the following methodological approaches are recommended:

Spectrophotometric NADPH Oxidation Assay:

• Reaction buffer: 100 mM potassium phosphate, pH 7.5 (for kidney enzyme) or pH optimum determined empirically for recombinant enzyme

• 0.1-0.2 mM NADPH as cofactor (obligatory requirement for enzyme function)

• PGE2 concentration range: 10-200 μM (consider Km value of 3.2 × 10⁻⁴ M for kidney enzyme)

• Monitor decrease in absorbance at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

• Temperature: 25°C or 37°C, with appropriate controls

• Include enzyme-free and substrate-free controls

Product Formation Assay:

• Incubate enzyme with PGE2 and NADPH under conditions above

• Extract products with ethyl acetate or solid-phase extraction

• Analyze PGF2α formation by HPLC, LC-MS, or immunoassay

• This approach confirms the actual conversion rather than just NADPH consumption

Alternative Substrate Assays:

• For characterizing the dual functionality of the enzyme:

  • 20α-hydroxyprogesterone formation from progesterone (20α-HSD activity)

  • Reduction of 9,10-phenanthrenquinone (shown active with bovine enzyme, Km = 6 μM)

  • Reduction of methylglyoxal and DL-glyceraldehyde

• These alternative substrates can provide complementary information about enzyme function

Activity Calculations and Reporting:

• Express activity as μmol product formed or NADPH consumed per minute per mg protein

• For kinetic parameters, use non-linear regression to fit to Michaelis-Menten equation

• Report both Vmax and Km values for each substrate tested

• For the bovine placental enzyme, Vmax was 183 pmol/min for PGE2

These assay conditions should be optimized for the recombinant enzyme, as properties may differ slightly from native enzyme due to expression system effects, presence of affinity tags, or differences in post-translational modifications.

How does substrate specificity compare between native and recombinant PGE2 9-reductase?

The substrate specificity of rabbit PGE2 9-reductase is remarkably broad, encompassing multiple classes of compounds. When characterizing recombinant enzyme, it's essential to compare its substrate profile with that of the native enzyme:

Primary Prostaglandin Activity:

• PGE2 to PGF2α conversion (defining activity)

• The native enzyme from kidney quantitatively converts PGE2 to PGF2α

• Recombinant enzyme should maintain this primary activity with similar efficiency

Steroid Hormone Metabolism:

• 20α-HSD activity (reduction of progesterone)

• Competitive inhibition between PGE2 and progesterone indicates they share the same binding site

• This dual functionality creates a direct link between prostaglandin and steroid metabolism pathways

Other Carbonyl Substrates:

• Aldehydes: methylglyoxal, DL-glyceraldehyde

• Quinones: 9,10-phenanthrenquinone

• The enzyme demonstrates wide substrate specificity for reducing various carbonyl compounds

Comparative Kinetic Parameters:
For proper comparison, kinetic parameters should be determined for multiple substrates:

The preservation of the dual catalytic activity (PGE2 9-reductase and 20α-HSD) is a critical quality control parameter for recombinant enzyme preparation. This unique characteristic distinguishes PGE2 9-reductase from many other enzymes and reflects its physiological significance in coordinating prostaglandin and steroid signaling pathways . Altered substrate preference in recombinant enzyme may indicate improper folding or structural differences from the native enzyme.

How can inhibition studies be designed to investigate the catalytic mechanism?

Inhibition studies provide valuable insights into the catalytic mechanism and substrate binding properties of recombinant rabbit PGE2 9-reductase. A comprehensive inhibition analysis should include:

Competitive Inhibition Analysis:

• The competitive inhibition between PGE2 and progesterone observed in the native enzyme provides a key starting point

• Design experiments with varying concentrations of both substrates to generate Dixon plots

• Determine Ki values for each substrate when functioning as an inhibitor

• This approach can confirm the shared binding site for prostaglandins and steroids in the recombinant enzyme

Cofactor Competition Studies:

• Test NADH versus NADPH to confirm cofactor specificity

• Determine if the strict NADPH dependence of the native enzyme is preserved in the recombinant form

• Calculate Km values for both cofactors to quantify preference

Structural Analog Inhibition:

• Test PGE1, PGE3, and other prostaglandin analogs as potential competitive inhibitors

• Examine structure-activity relationships using various steroid hormone analogs

• Use inhibition patterns to map the substrate binding pocket

Chemical Modification Studies:

• Employ group-specific reagents to identify essential catalytic residues:

  • Diethylpyrocarbonate (histidine modification)

  • Iodoacetamide (cysteine modification)

  • N-ethylmaleimide (thiol modification)

• Monitor activity loss to identify critical functional groups

pH-Dependent Inhibition:

• Perform inhibition studies across pH range (6.0-9.0)

• Identify ionizable groups involved in catalysis

• Compare with pH profile of kidney enzyme (broad profile with optimum at pH 7.5)

Data Analysis and Interpretation:

• Generate Lineweaver-Burk, Dixon, and Cornish-Bowden plots

• Determine inhibition type (competitive, non-competitive, uncompetitive)

• Calculate inhibition constants (Ki)

• Create mechanistic models explaining dual catalytic activity

These inhibition studies should help elucidate whether the recombinant enzyme maintains the same catalytic mechanism as the native enzyme, particularly the competitive relationship between prostaglandin and steroid substrates that suggests their binding to the same active site . This characteristic competitive inhibition pattern serves as an important validation criterion for properly folded recombinant enzyme.

How can site-directed mutagenesis be applied to investigate catalytic residues?

Site-directed mutagenesis represents a powerful approach for investigating the catalytic mechanism of recombinant rabbit PGE2 9-reductase. As a member of the aldo-keto reductase superfamily, specific residues can be targeted based on conserved sequences and catalytic mechanisms:

Target Residue Selection Strategy:

• Identify the catalytic triad residues (typically Tyr, Lys, Asp/Glu in aldo-keto reductases)

• Select NADPH-binding residues based on conserved motifs

• Target residues in substrate binding pocket that might confer dual specificity

• Choose residues based on sequence alignment with related enzymes and computational modeling

Experimental Design for Key Residues:

Dual Activity Investigation:

• Create mutations that selectively affect either PGE2 reductase or 20α-HSD activity

• Test if these activities can be uncoupled through strategic mutations

• Investigate the structural basis for the competitive inhibition between PGE2 and progesterone

Experimental Workflow:

• Generate mutants using overlap extension PCR or commercial site-directed mutagenesis kits

• Express and purify mutant proteins following protocols optimized for wild-type enzyme

• Characterize each mutant through:

  • Activity assays with multiple substrates

  • Determination of kinetic parameters (Km, kcat, kcat/Km)

  • pH-activity profiles

  • Inhibition studies

Data Interpretation Guidelines:

• Compare catalytic efficiency (kcat/Km) between wild-type and mutants

• Analyze changes in substrate preference ratios

• Assess pH-dependence profiles for mechanistic insights

• Correlate findings with structural models to develop comprehensive catalytic mechanism

This systematic mutagenesis approach will provide valuable information about the residues responsible for the unique dual functionality of rabbit PGE2 9-reductase and its classification as a member of the aldo-keto reductase superfamily . The findings can be correlated with the competitive inhibition observed between PGE2 and progesterone to develop a detailed understanding of how the same enzyme accommodates these structurally distinct substrates.

How can computational modeling enhance understanding of PGE2 9-reductase structure?

Computational modeling provides valuable tools for investigating the structure-function relationship of rabbit PGE2 9-reductase, particularly given its classification within the aldo-keto reductase superfamily and its dual catalytic activities:

Homology Modeling Approach:

• Select appropriate templates from the aldo-keto reductase superfamily with solved crystal structures

• Generate multiple models using tools like SWISS-MODEL, which has been successfully employed for similar enzymes

• Refine models through energy minimization and molecular dynamics simulations

• Validate models using Ramachandran plots, QMEAN scores, and comparison with experimental data

Substrate and Cofactor Binding Analysis:

• Dock NADPH to establish the cofactor binding site

• Perform comparative docking of PGE2 and progesterone to investigate dual substrate specificity

• Analyze the binding modes of alternative substrates (9,10-phenanthrenquinone, methylglyoxal)

• Identify key residues involved in substrate recognition and catalysis

Dual Activity Mechanism Investigation:

• Simulate enzyme conformational changes upon binding different substrates

• Analyze how the same active site accommodates structurally distinct substrates

• Investigate the molecular basis for the competitive inhibition between PGE2 and progesterone

• Predict residues that determine substrate preference

Integration with Experimental Approaches:

• Design site-directed mutagenesis experiments based on computational predictions

• Use models to interpret kinetic and inhibition data

• Guide protein engineering efforts to modify substrate specificity

• Assist in designing selective inhibitors for mechanistic studies

Advanced Computational Techniques:

• Molecular dynamics simulations to study protein flexibility and substrate access

• QM/MM methods to investigate the electronic aspects of the catalytic mechanism

• Free energy calculations to determine binding energy differences between substrates

• Consider applying modern neural network approaches like AlphaFold for improved structural prediction

The application of computational modeling represents an underutilized approach in recombinant enzyme research, with only 19% of studies employing bioinformatics and modeling tools . For rabbit PGE2 9-reductase, these techniques are particularly valuable for understanding how a single enzyme efficiently catalyzes reactions with structurally diverse substrates and the molecular mechanism behind the tight linking of prostaglandin and steroid metabolism observed in physiological contexts .

What structural features determine the dual catalytic activity of PGE2 9-reductase?

The unique dual catalytic activity of rabbit PGE2 9-reductase—functioning both as a PGE2 9-reductase and a 20 alpha-hydroxysteroid dehydrogenase—presents an intriguing structure-function relationship. Based on the available data, several structural features likely contribute to this dual functionality:

Active Site Architecture:

• The enzyme must possess a sufficiently spacious and flexible binding pocket to accommodate both the prostanoid ring structure of PGE2 and the steroid skeleton of progesterone

• The competitive inhibition observed between PGE2 and progesterone indicates they bind to the same active site

• Key hydrophobic residues likely create a versatile binding pocket that can adapt to different substrate shapes

Catalytic Residues:

• As a member of the aldo-keto reductase superfamily, the enzyme likely employs a conserved catalytic triad (Tyr, Lys, Asp/Glu)

• These residues must be positioned to catalyze the reduction of both the 9-keto group of PGE2 and the 20-keto group of progesterone

• The orientation of these catalytic residues likely allows the enzyme to accommodate different substrate binding modes

Cofactor Binding Domain:

• The strict NADPH dependence (rather than NADH) observed in both rabbit kidney and corpus luteum enzymes indicates a specific cofactor binding domain

• This domain must position the nicotinamide ring appropriately for hydride transfer to different substrate ketone groups

• The binding orientation ensures proper stereochemistry of the reduction

Substrate Specificity Determinants:

• The enzyme demonstrates broad substrate specificity beyond PGE2 and progesterone, including aldehydes, ketones, and quinones

• This suggests that the binding pocket recognizes carbonyl functionalities in diverse chemical contexts

• The varying affinity for different substrates (as indicated by the different Km values) suggests specific structural interactions that favor certain substrates

Evolutionary Significance:

• The dual activity creates a direct link between prostaglandin and steroid metabolism pathways

• This biochemical connection may have evolved to coordinate reproductive functions, particularly in regulating corpus luteum lifespan

• The enzyme could serve as a molecular switch between different signaling pathways

This structure-function relationship has significant physiological implications, particularly in reproductive tissues. The ability of PGE2 9-reductase to interconvert signaling molecules from different pathways positions it as a potential key regulator in the cascade of events leading to corpus luteum regression in rabbits . Understanding these structural determinants could lead to the development of selective modulators for reproductive research and potentially therapeutic applications.

How can recombinant PGE2 9-reductase be used to investigate corpus luteum function?

Recombinant rabbit PGE2 9-reductase provides a valuable research tool for investigating corpus luteum (CL) function and the molecular mechanisms governing its lifespan. Several experimental approaches can be implemented:

Temporal Expression Studies:

• Use recombinant enzyme as a standard for quantifying native enzyme levels

• Monitor PGE2 9-reductase expression throughout the CL lifespan in pregnant versus pseudopregnant rabbits

• Correlate enzyme activity with the observed significant differences between pregnancy and pseudopregnancy, particularly on Days 10-15

• This approach can clarify the role of PGE2 9-reductase in determining CL persistence or regression

PGE2/PGF2α Ratio Manipulation:

• Use controlled amounts of recombinant enzyme in ex vivo CL tissue cultures

• Manipulate local PGE2/PGF2α ratios through enzyme addition

• Monitor effects on progesterone production and apoptotic markers

• This can test the hypothesis that PGE2 9-reductase regulates CL lifespan by modulating the balance between luteotropic PGE2 and luteolytic PGF2α

Dual Activity Investigation:

• Explore how the dual PGE2 9-reductase and 20α-HSD activities coordinate prostaglandin and steroid metabolism

• Determine if these activities are differentially regulated during various reproductive states

• Investigate how competitive substrate inhibition between PGE2 and progesterone influences local hormone concentrations

Interventional Studies:

• Develop specific inhibitors based on recombinant enzyme studies

• Apply these inhibitors in vivo to manipulate enzyme activity at specific timepoints

• Monitor effects on CL lifespan, progesterone production, and pregnancy outcomes

• This approach can establish causality between enzyme activity and physiological outcomes

Gene Expression Regulation:

• Use insights from recombinant enzyme studies to investigate transcriptional and post-translational regulation

• Identify factors that control the temporal patterns of enzyme activity

• Study how hormone signaling pathways converge to regulate PGE2 9-reductase expression

These research applications directly address the enzyme's proposed role as "a key enzyme in the cascade of events leading to the regression of the corpus luteum in the rabbit" . By manipulating this enzymatic activity, researchers can gain deeper insights into the molecular mechanisms controlling reproductive cyclicity and potentially develop new approaches for managing fertility.

What are the challenges in developing specific inhibitors for PGE2 9-reductase?

Developing specific inhibitors for rabbit PGE2 9-reductase presents several challenges that must be addressed through systematic research approaches:

Dual Activity Selectivity:

• The enzyme's dual functionality (PGE2 9-reductase and 20α-HSD) complicates inhibitor design

• Determining whether to target both activities or selectively inhibit one function

• Need to understand if these activities share the same active site but with different substrate orientations

• Competitive relationship between PGE2 and progesterone must be considered in inhibitor design

Structural Homology Challenges:

• PGE2 9-reductase belongs to the aldo-keto reductase superfamily with many homologous enzymes

• Achieving specificity against related enzymes requires detailed structural knowledge

• Risk of off-target effects on other NADPH-dependent reductases

• Need to identify unique structural features that distinguish rabbit PGE2 9-reductase

Tissue Specificity Considerations:

• Different properties between corpus luteum and kidney enzymes (36 kDa vs 21.8 kDa)

• Deciding whether to target all tissue variants or specific isoforms

• Understanding if inhibitors will have tissue-specific effects

• Potential for different inhibitor sensitivities between tissue variants

Methodological Approach for Inhibitor Development:

• Structure-Based Design:

  • Generate computational models of enzyme based on aldo-keto reductase family structures

  • Perform virtual screening of compound libraries targeting the active site

  • Design transition state analogs that mimic the reaction intermediate

• Substrate-Based Design:

  • Develop modified PGE2 or progesterone analogs that bind but resist catalysis

  • Create hybrid molecules incorporating features of both substrate classes

  • Exploit the competitive relationship between substrates

• High-Throughput Screening:

  • Establish robust activity assays suitable for screening compound libraries

  • Screen natural product libraries for lead compounds

  • Implement counter-screens against related enzymes to assess specificity

• Validation Strategy:

  • Test inhibitor effects on both PGE2 9-reductase and 20α-HSD activities

  • Determine inhibition mechanisms and constants

  • Evaluate specificity against related aldo-keto reductases

  • Assess cellular and tissue effects in reproductive models

Developing such inhibitors would provide valuable research tools for investigating the physiological significance of PGE2 9-reductase in corpus luteum function and potentially lead to novel approaches for modulating reproductive processes.

How can recombinant PGE2 9-reductase contribute to comparative studies across species?

Recombinant rabbit PGE2 9-reductase offers an excellent platform for comparative enzymology studies across species, providing insights into evolutionary adaptation and species-specific reproductive physiology:

Cross-Species Enzyme Characterization:

• Compare rabbit enzyme properties with bovine placental PGE2 9-reductase (45 kDa)

• Examine differences in kinetic parameters between species:

  • Rabbit kidney: Km for PGE2 = 3.2 × 10⁻⁴ M

  • Bovine placenta: Km for PGE2 = 117 μM

• Investigate differences in molecular weight across species and tissues:

  • Rabbit corpus luteum: 36 kDa

  • Rabbit kidney: 21.8 kDa

  • Bovine placenta: 45 kDa

Evolutionary Analysis:

• Perform phylogenetic analysis of PGE2 9-reductase across mammalian species

• Identify conserved catalytic residues versus variable substrate-binding regions

• Correlate enzyme properties with species-specific reproductive strategies

• Investigate how structural variations relate to differences in reproductive physiology

Reproductive Physiology Variations:

• Compare enzyme activity patterns during pregnancy across species

• Examine species differences in corpus luteum regression mechanisms

• Investigate how the PGE2/PGF2α balance is regulated in different mammals

• Study the relationship between enzyme activity and gestation length/implantation timing

Methodological Advantages:

• Recombinant expression provides standardized enzymes for direct comparisons

• Eliminates variability associated with tissue extraction and purification

• Allows for creation of chimeric enzymes to identify species-specific functional domains

• Enables site-directed mutagenesis to convert properties between species

Translational Applications:

• Identify species-specific properties relevant to reproductive biotechnology

• Develop targeted approaches for fertility control in various species

• Apply insights to conservation efforts for endangered species

• Translate findings to veterinary and potentially human reproductive medicine

This comparative approach using recombinant enzymes can reveal how PGE2 9-reductase has evolved to meet the specific reproductive requirements of different species. The variations in enzyme properties between rabbit tissues (corpus luteum vs. kidney) and between species (rabbit vs. bovine) suggest significant evolutionary adaptation of this enzyme to fulfill species-specific and tissue-specific functions in prostaglandin metabolism and reproductive physiology.