title: Disruption of the proton relay network in the class 2 dihydroorotate dehydrogenase from Escherichia coli.
abstract: Dihydroorotate dehydrogenases (DHODs) are FMN-containing enzymes that catalyze the conversion of dihydroorotate (DHO) to orotate in the de novo synthesis of pyrimidines. During the reaction, a proton is transferred from C5 of DHO to an active site base and the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of the flavin as a hydride. In class 2 DHODs, a hydrogen bond network observed in crystal structures has been proposed to deprotonate the C5 atom of DHO. The active site base (Ser175 in the Escherichia coli enzyme) hydrogen bonds to a crystallographic water molecule that sits on a phenylalanine (Phe115 in the E. coli enzyme) and hydrogen bonds to a threonine (Thr178 in the E. coli enzyme), residues that are conserved in class 2 enzymes. The importance of these residues in the oxidation of DHO was investigated using site-directed mutagenesis. Mutating Ser175 to alanine had severe effects on the rate of flavin reduction, slowing it by more than 3 orders of magnitude. Changing the size and/or hydrophobicity of the residues of the hydrogen bond network, Thr178 and Phe115, slowed flavin reduction as much as 2 orders of magnitude, indicating that the active site base and the hydrogen bond network work together for efficient deprotonation of DHO.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-5606, USA.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Kow
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Whicher
authors.forename: Claudia A
authors.initials: CA
authors.lastname: McDonald
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Palfey
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Fagan
mesh_terms: D020134:Catalytic Domain; D000090722:Dihydroorotate Dehydrogenase; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D029968:Escherichia coli Proteins; D005486:Flavin Mononucleotide; D044925:Oxidoreductases Acting on CH-CH Group Donors
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