Dihydroorotate dehydrogenase B of Enterococcus faecalis. Characterization and insights into chemical mechanism.
Enterococcus faecalis dihydroorotate dehydrogenase B is a heterodimer of 28 and 33 kDa encoded by the pyrK and pyrDb genes. Both subunits copurify during all chromatographic steps, and, as determined by HPLC, one FMN and one FAD are bound per heterodimer. The enzyme catalyzes efficient oxidation of 4-S-NADH by orotate. Isotope effect and pH data suggest that reduction of flavin by NADH at the PyrK site is only partially rate limiting with no kinetically significant proton transfer occurring in the reductive half-reaction; therefore, a group exhibiting a pK of 5.7 +/- 0.2 represents a residue involved in binding of NADH rather than in catalysis. The reducing equivalents are shuttled between the NADH-oxidizing flavin in PyrK and the orotate-reacting flavin in PyrDb, by iron-sulfur centers through flavin semiquinones as intermediates. A solvent kinetic isotope effect of 2.5 +/- 0.2 on V is indicative of rate-limiting protonation in the oxidative half-reaction and most likely reflects the interaction between the isoalloxazine N1 of the orotate-reducing flavin and Lys 168 (by analogy with L. lactis DHODase A). The oxidative half-reaction is facilitated by deprotonation of the group(s) with pK(s) of 5.8-6.3 and reflects either deprotonation of the reduced flavin or binding of orotate; this step is followed by hydride transfer to C6 and general acid-assisted protonation (pK of 9.1 +/- 0.2) at C5 of the product.
Marcinkeviciene
J
J
Department of Chemical Enzymology and Antimicrobial Group, DuPont Pharmaceuticals, Wilmington, Delaware 19880-0400, USA.
Tinney
L M
LM
Wang
K H
KH
Rogers
M J
MJ
Copeland
R A
RA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D003903:Deuterium; D000090722:Dihydroorotate Dehydrogenase; D013293:Enterococcus faecalis / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000737:chemistry; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005415:Flavins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D012997:Solvents; D013053:Spectrophotometry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D000090722:Dihydroorotate Dehydrogenase; D005415:Flavins; C100899:PyrK protein, Lactococcus lactis; D012997:Solvents; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D003903:Deuterium; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors