Molecular Structure of Dihydroorotase:  A Paradigm for Catalysis through the Use of a Binuclear Metal Center

• Published in: Biochemistry (Easton) 2001-06, Vol.40 (24), p.6989-6997
• Main Authors: Thoden, James B, Phillips, George N, Neal, Tamiko M, Raushel, Frank M, Holden, Hazel M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Aryldialkylphosphatase; Aspartic Acid - chemistry; Binding Sites; Carbamyl Phosphate - chemistry; Catalysis; Crystallography, X-Ray; dihydroorotase; Dihydroorotase - chemistry; Dihydroorotase - metabolism; Dimerization; Escherichia coli; Escherichia coli - enzymology; Esterases - chemistry; Humans; Lysine - chemistry; Molecular Sequence Data; Orotic Acid - analogs & derivatives; Orotic Acid - metabolism; pyrimidines; Structure-Activity Relationship; Zinc - chemistry; Zinc - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010682i

Dihydroorotase plays a key role in pyrimidine biosynthesis by catalyzing the reversible interconversion of carbamoyl aspartate to dihydroorotate. Here we describe the three-dimensional structure of dihydroorotase from Escherichia coli determined and refined to 1.7 Å resolution. Each subunit of the homodimeric enzyme folds into a “TIM” barrel motif with eight strands of parallel β-sheet flanked on the outer surface by α-helices. Unexpectedly, each subunit contains a binuclear zinc center with the metal ions separated by ∼3.6 Å. Lys 102, which is carboxylated, serves as a bridging ligand between the two cations. The more buried or α-metal ion in subunit I is surrounded by His 16, His 18, Lys 102, Asp 250, and a solvent molecule (most likely a hydroxide ion) in a trigonal bipyramidal arrangement. The β-metal ion, which is closer to the solvent, is tetrahedrally ligated by Lys 102, His 139, His 177, and the bridging hydroxide. l-Dihydroorotate is observed bound to subunit I, with its carbonyl oxygen, O4, lying 2.9 Å from the β-metal ion. Important interactions for positioning dihydroorotate into the active site include a salt bridge with the guanidinium group of Arg 20 and various additional electrostatic interactions with both protein backbone and side chain atoms. Strikingly, in subunit II, carbamoyl l-aspartate is observed binding near the binuclear metal center with its carboxylate side chain ligating the two metals and thus displacing the bridging hydroxide ion. From the three-dimensional structures of the enzyme-bound substrate and product, it has been possible to propose a unique catalytic mechanism for dihydroorotase. In the direction of dihydroorotate hydrolysis, the bridging hydroxide attacks the re-face of dihydroorotate with general base assistance by Asp 250. The carbonyl group is polarized for nucleophilic attack by the bridging hydroxide through a direct interaction with the β-metal ion. During the cyclization of carbamoyl aspartate, Asp 250 initiates the reaction by abstracting a proton from N3 of the substrate. The side chain carboxylate of carbamoyl aspartate is polarized through a direct electrostatic interaction with the binuclear metal center. The ensuing tetrahedral intermediate collapses with C−O bond cleavage and expulsion of the hydroxide which then bridges the binuclear metal center.