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 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).
Neidhardt
E A
EA
Procept, Inc., Cambridge, MA 02139, USA.
Punreddy
S R
SR
McLean
J E
JE
Hedstrom
L
L
Grossman
T H
TH
D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D000090722:Dihydroorotate Dehydrogenase; D004926:Escherichia coli; D006639:Histidine; D006801:Humans; D007165:Immunosuppression Therapy; D007700:Kinetics; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D012995:Solubility; D013347:Subcellular Fractions; D014451:Ubiquinone / Q000378:metabolism
D000090722:Dihydroorotate Dehydrogenase; D011993:Recombinant Fusion Proteins; D014451:Ubiquinone; C004768:4,5-dihydroorotic acid; D006639:Histidine; D009963:Orotic Acid; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors