Roles in Binding and Chemistry for Conserved Active Site Residues in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli

Abstract

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 E. 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 is 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 are 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 build-up in the active site. This finding is consistent with previous results that Class 2 DHODs use a stepwise mechanism for DHO oxidation.Dihydroorotate dehydrogenases (DHODs 1 ) are flavin-containing enzymes that catalyze the fourth step (the only redox step) in the de novo synthesis of pyrimidines -the conversion of dihydroorotate (DHO) to orotate (OA) (Scheme 1). DHODs have been categorized into two broad classes based on sequence (1). Several properties of the enzymes segregate nicely into these classes. Class 2 DHODs are membrane-bound enzymes that are oxidized by ubiquinone (2). Class 1 enzymes are cytosolic proteins that have been further divided into two subclasses. Class 1A enzymes are homodimers that are oxidized by fumarate (3). Class 1B enzymes are α 2 b 2 heterotetramers that contain a subunit that resembles the Class 1A enzymes and a second subunit that contains FAD and an iron-sulfur cluster, allowing the enzyme to be oxidized by NAD (4). Class 1 DHODs are found mostly in Gram-positive bacteria, although a few microbial eukaryotes also have Class 1A DHODs, while Class 2 enzymes are found in Gram-negative bacteria and almost all eukaryotes.*To whom correspondence should be addressed at: Department of Biological Chemistry, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, Phone: (734) 615-2452; Fax: (734) The catalytic cycle of DHODs can be studied in two separate half-reactions, allowing the direct observation of the chemistry. The reductive half-reaction involves the oxidation of DHO to OA with the concomitant reduction of the enzyme-bound FMN. The kinetics of the reductive half-reaction has been studied in anaerobic stopped-flow experiments for several Class 2 ...

Pyrimidine Pathway-Dependent and -Independent Functions of the Toxoplasma gondii Mitochondrial Dihydroorotate Dehydrogenase

“…Enzyme activity was not detected in the double mutant. Overall, these impaired kinetic properties of TgDHODH activity are consistent with previous mutagenesis studies performed on corresponding mutations developed in the E. coli DHODH (31) and reinforce the importance of these key asparagine residues in catalysis. TgDHODH differs from other family 2 DHODHs in containing an extended N-terminal targeting sequence of 157 amino acids that appears to be cleaved during mitochondrial import (22).…”

“…Attempts to obtain a detectable activity for the DM by increasing enzyme and substrate concentrations were unsuccessful. These results correlate with defects in kinetic parameters previously observed in the corresponding mutant E. coli DHODH enzymes (31), reinforcing the critical importance of these asparagine residues in stabilizing the pyrimidine for efficient catalytic activity of family 2 DHODH enzymes.…”

“…DHODH oxidizes dihydroorotate to orotate with coordinate reduction of the enzymebound FMN in a stepwise reaction. Site-directed mutagenesis of several conserved asparagine (N) residues in E. coli DHODH adversely affected substrate or product binding as well as reduction rate constants (31). Mutation of N172 of E. coli DHODH caused a large decrease in the reduction rate constant with a slight decrease in dihydroorotate affinity, while mutation of N246 caused a large decrease in both kinetic parameters (31).…”

“…Site-directed mutagenesis of several conserved asparagine (N) residues in E. coli DHODH adversely affected substrate or product binding as well as reduction rate constants (31). Mutation of N172 of E. coli DHODH caused a large decrease in the reduction rate constant with a slight decrease in dihydroorotate affinity, while mutation of N246 caused a large decrease in both kinetic parameters (31). Examination of the E. coli DHODH crystallographic structure shows that residues N172 and N246 are predicted to establish three hydrogen bonds with orotate ( Fig.…”

Insights into the mechanism of oxidation of dihydroorotate to orotate catalysed by human class 2 dihydroorotate dehydrogenase: a QM/MM free energy study

“…The mechanism of the dehydrogenation of dihydroorotic acid 50 by DHOD has been the subject of extensive studies 2, 9,10,[12][13][14][15][16][17][18][19][20] . The oxidation reaction of DHO breaks two carbon-hydrogen bonds.…”