Recombinant Human Dihydroorotate Dehydrogenase Protein, CF

Catalog # Availability Size / Price Qty
10062-DD-020
DHODH Protein Enzyme Activity
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Product Details
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Recombinant Human Dihydroorotate Dehydrogenase Protein, CF Summary

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.
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.
Source
E. coli-derived human DHODH protein
Thr31 - Arg395
with N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
41 kDa
SDS-PAGE
41 kDa, reducing conditions

Product Datasheets

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.

Assay Procedure

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

Data Images

Binding Activity DHODH Protein Enzyme Activity View Larger

Recombinant Human DHODH (Catalog # 10062-DD) is measured byits reduction of 2,6-dichloroindophenol during oxidation of dihydroorotate.

SDS-PAGE DHODH Protein Enzyme Activity View Larger

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

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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
Entrez Gene IDs
1723 (Human); 56749 (Mouse); 65156 (Rat)
Alternate Names
DHOdehase; DHODH; dihydroorotate dehydrogenase; dihydroorotate dehydrogenase, mitochondrial; Dihydroorotate Oxidase; EC 1.3.3.1; EC 1.3.5.2; human complement of yeast URA1; POADS; URA1

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