DHFR Mouse
Description
Enzyme Function and Mechanism
Mouse DHFR catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as an electron donor . This reaction is critical for synthesizing purines, thymidylic acid, and methionine, supporting DNA replication and one-carbon metabolism .
Key Features:
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Active Site: Conformational flexibility of the Met20 loop stabilizes NADPH and facilitates hydride transfer .
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pH Sensitivity: The hydride transfer step is pH-dependent, influenced by protonation and water-mediated interactions .
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Structural Domains: Contains an 8-stranded β-pleated sheet core with α-helices connecting β-strands .
Comparative Analysis of DHFR Isoforms
Mouse DHFR belongs to the class I family, distinct from bacterial type II enzymes like R67 DHFR.
Knockout and Mutant Models
Dhfr Knockout Mice
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Phenotype: Embryonic lethality in homozygotes; heterozygotes viable but exhibit reduced DHFR activity (50% of WT) .
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Pathological Features:
Dhfr Mutant (Thr136Ala)
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Observations: Hematopoiesis arrest and vascular relaxation defects linked to altered DHFR activity .
Transgenic Models
Endothelial-Specific DHFR Overexpression (tg-EC-DHFR)
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PH Protection: Reverses DAHP- or hypoxia-induced PH via restored NO bioavailability and reduced ROS .
Source and Purification
| Parameter | Value |
|---|---|
| Source | E. coli (His-tagged) |
| Molecular Weight | 23.8 kDa (207 aa) |
| Purity | >95% (SDS-PAGE) |
| Activity | >0.2 units/mg (pH 6.5, 25°C) |
Applications:
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Biochemical Assays: Substrate specificity studies (e.g., methotrexate resistance) .
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Therapeutic Research: Testing DHFR-targeted anticancer agents .
Neurogenesis Regulation
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Role in Neocortex Development: DHFR deficiency reduces SAM levels, impairing H3K4 trimethylation and indirect neurogenesis .
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Mechanistic Insight: DHFR activity is rate-limiting for methionine cycle flux in neural progenitors .
Pulmonary Hypertension
| Model | mPAP (mmHg) | RVSP (mmHg) | Medial Thickness |
|---|---|---|---|
| WT | 15.2 ± 0.5 | 19.8 ± 0.6 | 25.9% (vessels <200 µm) |
| DHFR KO | 28.4 ± 1.2 | 31.7 ± 1.1 | 47.95% (vessels <200 µm) |
| tg-EC-DHFR | 16.1 ± 0.7 | 20.3 ± 0.8 | 26.4% (vessels <200 µm) |
Key Findings:
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DHFR deficiency causes eNOS uncoupling, increased oxidative stress, and vascular remodeling .
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DHFR overexpression reverses PH by restoring NO/tetrahydrobiopterin (BH4) balance .
Antibiotic Resistance Studies
Product Specs
Product Science Overview
DHFR is a small enzyme, approximately 21 kDa in size . It is highly conserved across different species, including humans and mice. The enzyme’s primary function is to maintain the cellular levels of tetrahydrofolate, a cofactor required for one-carbon transfer reactions . These reactions are crucial for the synthesis of nucleotides and amino acids, making DHFR indispensable for cell growth and division .
Recombinant DHFR refers to the enzyme produced through recombinant DNA technology. This involves inserting the DHFR gene into a suitable expression system, such as E. coli, to produce the enzyme in large quantities . Recombinant DHFR is often tagged with a histidine tag to facilitate purification and is used in various biochemical assays and research applications .
Recombinant DHFR is widely used in research to study its role in cellular metabolism and its potential as a target for chemotherapy drugs. Inhibitors of DHFR, such as methotrexate, are used to treat certain types of cancer and autoimmune diseases by blocking the enzyme’s activity, thereby inhibiting DNA synthesis and cell proliferation .
The study of DHFR, particularly its recombinant forms, is crucial in drug development. By understanding how DHFR functions and how it can be inhibited, researchers can develop more effective drugs with fewer side effects. The enzyme’s role in folate metabolism also makes it a target for antibiotics and antimalarial drugs .
