Roles in binding and chemistry for conserved active site residues in the class 2 dihydroorotate dehydrogenase from Escherichia coli.
Dihydroorotate dehydrogenases (DHODs) catalyze the only redox step in de novo pyrimidine biosynthesis, the oxidation of dihydroorotate (DHO) to orotate (OA). During the reaction, the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of an enzyme-bound FMN prosthetic group as a hydride, and an active site base (Ser175 in the class 2 DHOD from Escherichia coli) deprotonates C5 of DHO. Aside from the identity of the active site base, the pyrimidine binding site of all DHODs is nearly identical. Several strictly conserved residues (four asparagines and either a serine or threonine) make extensive hydrogen bonds to the pyrimidine). The roles these conserved residues play in DHO oxidation are unknown. Site-directed mutagenesis was used to investigate the role of each residue during DHO oxidation. The effects of each mutation on substrate and product binding, as well as the effect on the rate constant of the chemical step, were determined. The effects of the mutations ranged from negligible to severe. Some of the residues were very important for chemistry, while others were important for binding. Mutation of residues capable of stabilizing reaction intermediates resulted in large decreases in the rate constant of the chemical step, suggesting these residues are quite important for stabilizing charge buildup in the active site. This finding is consistent with previous results that class 2 DHODs use a stepwise mechanism for DHO oxidation.
Fagan
Rebecca L
RL
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-5606, USA.
Palfey
Bruce A
BA
D020134:Catalytic Domain; D000090722:Dihydroorotate Dehydrogenase; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
D000090722:Dihydroorotate Dehydrogenase; D029968:Escherichia coli Proteins; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; D044925:Oxidoreductases Acting on CH-CH Group Donors