Expression and characterization of E. coli-produced soluble, functional human dihydroorotate dehydrogenase: a potential target for immunosuppression

Human dihydroorotate dehydrogenase (huDHODH) is essential for de novo biosynthesis of pyrimidines and the target of two immunosuppressive drugs, brequinar and the leflunomide metabolite A77-1726 (Chen et al., 1992; Davis et al., 1996). Using a T7 RNA polymerase expression system, we produced huDHODH...

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Published inJournal of molecular microbiology and biotechnology Vol. 1; no. 1; p. 183
Main Authors Neidhardt, E A, Punreddy, S R, McLean, J E, Hedstrom, L, Grossman, T H
Format Journal Article
LanguageEnglish
Published Switzerland 01.08.1999
Subjects
Animals
Cattle
Cloning, Molecular
Escherichia coli
Histidine
Humans
Immunosuppression
Kinetics
Orotic Acid - analogs & derivatives
Orotic Acid - metabolism
Oxidoreductases - genetics
Oxidoreductases - isolation & purification
Oxidoreductases - metabolism
Oxidoreductases - physiology
Oxidoreductases Acting on CH-CH Group Donors
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - isolation & purification
Recombinant Fusion Proteins - metabolism
Recombinant Fusion Proteins - physiology
Solubility
Subcellular Fractions
Ubiquinone - metabolism
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Summary:Human dihydroorotate dehydrogenase (huDHODH) is essential for de novo biosynthesis of pyrimidines and the target of two immunosuppressive drugs, brequinar and the leflunomide metabolite A77-1726 (Chen et al., 1992; Davis et al., 1996). Using a T7 RNA polymerase expression system, we produced huDHODH as a fusion protein containing an amino-terminal decahistidine tag. Escherichia coli growth and expression conditions were optimized to enhance huDHODH solubility and to permit purification of the enzyme in the absence of detergent. Soluble huDHODH, purified by a simple two-step procedure, was catalytically active, monomeric, and contained a flavin mononucleotide (FMN) cofactor in a 1:1 FMN/protein molar ratio. Kinetic analysis showed that huDHODH uses a two site ping-pong mechanism, where DHO is oxidized at one site and the second substrate, ubiquinone, is reduced at the other. This result is consistent with the mechanism proposed for bovine liver DHODH (Hines and Johnston, 1989).
ISSN:1464-1801