A second dihydroorotate dehydrogenase (Type A) of the human pathogen Enterococcus faecalis: expression, purification, and steady-state kinetic mechanism.
We report the identification, expression, and characterization of a second Dihydroorotate dehydrogenase (DHODase A) from the human pathogen Enterococcus faecalis. The enzyme consists of a polypeptide chain of 322 amino acids that shares 68% identity with the cognate type A enzyme from the bacterium Lactococcus lactis. E. faecalis DHODase A catalyzed the oxidation of l-dihydroorotate while reducing a number of substrates, including fumarate, coenzyme Q(0), and menadione. The steady-state kinetic mechanism has been determined with menadione as an oxidizing substrate at pH 7.5. Initial velocity and product inhibition data suggest that the enzyme follows a two-site nonclassical ping-pong kinetic mechanism. The absorbance of the active site FMN cofactor is quenched in a concentration-dependent manner by titration with orotate and barbituric acid, two competitive inhibitors with respect to dihydroorotate. In contrast, titration of the enzyme with menadione had no effect on FMN absorbance, consistent with nonoverlapping binding sites for dihyroorotate and menadione, as suggested from the kinetic mechanism. The reductive half-reaction has been shown to be only partially rate limiting, and an attempt to evaluate the slow step in the overall reaction has been made by simulating orotate production under steady-state conditions. Our data indicate that the oxidative half-reaction is a rate-limiting segment, while orotate, most likely, retains significant affinity for the reduced enzyme, as suggested by the product inhibition pattern.
Marcinkeviciene
J
J
Department of Chemical Enzymology, DuPont Pharmaceutical Co., Wilmington, Delaware 19880-0400, USA. Jovita.Marcinkeviciene@dupontpharma.com
Jiang
W
W
Locke
G
G
Kopcho
L M
LM
Rogers
M J
MJ
Copeland
R A
RA
D000595:Amino Acid Sequence; D001463:Barbiturates / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D003001:Cloning, Molecular; D000090722:Dihydroorotate Dehydrogenase; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005650:Fumarates / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D017186:Titrimetry; D014812:Vitamin K / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology
D001463:Barbiturates; D000090722:Dihydroorotate Dehydrogenase; D005650:Fumarates; D011994:Recombinant Proteins; D014812:Vitamin K; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; C032005:fumaric acid; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010100:Oxygen; C032232:barbituric acid