Recombinant Human Dihydroorotate Dehydrogenase Protein, CF

R&D Systems | Catalog # 10062-DD

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human Dihydroorotate Dehydrogenase Protein (10062-DD)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

Q02127

Applications

Enzyme Activity
View all DHODH Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human DHODH protein
Thr31 - Arg395
with N-terminal Met and 6-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

41 kDa

SDS-PAGE

41 kDa, reducing conditions

Activity

Measured by its reduction of 2,6-dichloroindophenol during the oxidation of dihydroorotate.
The specific activity is >15000 pmol/min/μg, as measured under the described conditions.

Scientific Data Images for Recombinant Human Dihydroorotate Dehydrogenase Protein, CF

Recombinant Human Dihydroorotate Dehydrogenase Protein Binding Activity

Recombinant Human Dihydroorotate Dehydrogenase Protein Binding Activity

Recombinant Human DHODH (Catalog # 10062-DD) is measured by its reduction of 2,6-dichloroindophenol during oxidation of dihydroorotate.
Recombinant Human Dihydroorotate Dehydrogenase Protein SDS-PAGE

Recombinant Human Dihydroorotate Dehydrogenase Protein SDS-PAGE

2 μg/lane of Recombinant Human DHODH was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band at 41 kDa.

Formulation, Preparation, and Storage

10062-DD
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: DHODH

Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme in the pyrimidine de novo pathway that converts dihydroorotate to orotate. DHODHs have a monomeric structure composed of a large C-terminal a/b barrel and a small N-terminal helical domain (1). There are two main classes of DHODHs based on similarity, preference of substrate, and subcellular location (1,2). Within Class 2, structural differences can be exploited for selective targeting (3,4).  Human DHODH belongs to Class 2 and is a monomeric, mitochondrial, FMN-dependent enzyme (2). It is ubiquitously expressed in most tissue. As DHODH catalyzes de novo pyrimidine synthesis for synthesis of DNA/RNA essential to rapidly proliferating cells, DHODH is currently a target for treatment of cancer (5-8). It has also been successfully targeted in treatment for rheumatoid arthritis (9-10), multiple sclerosis (11-12), viral infection (13-14), microbial infectious diseases such as malaria (4, 15) and gastrointestinal disease (3, 16), and antifungal infection (17). Mutations in the DHODH resulting in functional defects cause Miller syndrome, also known as postaxial acrofacial dystosis syndrome (POADS) (18).

References

  1. Liu, S. et al. (2000) Structure. 8:25.
  2. Reis, R. A. G. et al. (2017) Arch. Biochem. Biophys. 632:175.
  3. Copeland, R. A. et al. (2000) J. Biol. Chem. 275:33373.
  4. Singh, A. et al. (2017) Eur. J. Med. Chem. 125:640.
  5. Baumann, P. et al. (2009) Mol. Cancer Ther. 8:366.
  6. Zhu, S. et al. (2013) PLoS One. 8:e71555.
  7. Lewis, T. A. et al. (2016) ACS Med. Chem. Lett. 7:1112.
  8. Koundinya, M. et al. (2018) Cell Chem. Biol. 21:705.
  9. Herrmann, M. L. et al. (2000) Immunopharmacology. 47:273.
  10. Li, E. K. et al. (2004) Clin. Ther. 26:447.
  11. Warnke, C. et al. (2013) Clin. Neurol. Neurosurg. 115:S90.
  12. O'Connor, P. et al. (2016) Neurology 86:920.
  13. Wang, Q. Y. et al. (2011) J. Virol. 85:6548.
  14. Smee, D. F. et al. (2012) Antivir. Chem. Chemother. 22:263.
  15. Skerlj, R. T. et al. (2011) ACS Med. Chem. Lett. 2:708.
  16. Ohishi, T. et al. (2018) Helicobacter 23:e12470.
  17. Oliver, J. D. et al. (2016) Proc. Natl. Acad. Sci. USA 113:12809.
  18. Fang, J. et al. (2012) Biosci. Rep. 32:631.

Long Name

Dihydroorotate Dehydrogenase

Alternate Names

DHOdehase, Dihydroorotate Oxidase, POADS, URA1

Entrez Gene IDs

1723 (Human); 56749 (Mouse); 65156 (Rat)

Gene Symbol

DHODH

UniProt

Q02127

Additional DHODH Products

Product Documents for Recombinant Human Dihydroorotate Dehydrogenase Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human Dihydroorotate Dehydrogenase Protein, CF

For research use only

Related Research Areas

Redox Enzymes

Citations for Recombinant Human Dihydroorotate Dehydrogenase Protein, CF

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Protocols

View specific protocols for Recombinant Human Dihydroorotate Dehydrogenase Protein, CF (10062-DD):

Materials
  • Assay Buffer: 50 mM Tris, 150 mM KCl, 0.1% Triton® X-100, pH 8.0
  • Recombinant Human DHODH His-tag (rhDHODH) (Catalog # 10062-DD)
  • L-Dihydroorotic acid (Sigma, Catalog # D7128), 40 mM stock in DMF
  • Decylubiquinone (Sigma, Catalog # D7911), 20 mM stock in DMSO
  • 2,6-Dichloroindophenol sodium salt hydrate (DPIP) (Sigma, Catalog # D1878), 2 mM in Absolute Ethanol
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhDHODH to 0.4 µg/mL in Assay Buffer.
  2. Prepare Substrate Mixture containing 2 mM L-Dihydroorotic acid, 0.2 mM Decylubiquinone and 0.12 mM DPIP in Assay Buffer.
  3. Load 50 µL of 0.4 µg/mL rhDHODH to plate, and start the reaction by adding 50 µL of Substrate Mixture.  Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
  4. Immediately read plate in kinetic mode for 5 minutes at 600 nm (absorbance).
  5. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Adjusted Vmax* (OD/min) x (-1) x well volume (L) x 1012 pmol/mol
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)

*Adjusted for Substrate Blank
**Using the extinction coefficient 21,000 M-1 cm-1.
***Using the path correction 0.32 cm.
Note: the output of many spectrophotometers is in mOD.

Per Well:

  • rhDHODH: 0.02 µg
  • L-Dihydroorotic acid: 1 mM
  • Decylubiquinone: 0.1 mM
  • DPIP: 0.06 mM

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