Molecular structure of dihydroorotase: A paradigm for catalysis through the use of a binuclear metal center

Dihydroorotase plays a key role in pyrimidine biosynthesis by catalyzing th e reversible interconversion of carbamoyl aspartate to dihydroorotate. Here we describe the three-dimensional structure of dihydroorotase from Escheri chia coil determined and refined to 1.7 Angstrom resolution. Each subunit o f the homodimeric enzyme folds into a "TIM" barrel motif with eight strands of parallel beta -sheet flanked on the outer surface by alpha -helices. Un expectedly, each subunit contains a binuclear zinc center with the metal io ns separated by similar to3.6 Angstrom. Lys 102, which is carboxylated, ser ves as a bridging ligand between the two cations. The more buried or a-meta l 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 a rrangement. The beta -metal ion, which is closer to the solvent, is tetrahe drally 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 Angstrom from the beta -metal ion. Important interactions for p ositioning dihydroorotate into the active site include a salt bridge with h e guanidinium group of Arg 20 and various additional electrostatic interact ions with both protein backbone and side chain atoms. Strikingly, in subuni t II, carbamoyl L-aspartate is observed binding near the binuclear metal ce nter with its carboxylate side chain ligating the two metals and thus displ acing the bridging hydroxide ion. From the three-dimensional structures of the enzyme-bound substrate and product, it has been possible to propose a u nique catalytic mechanism for dihydroorotase. In the direction of dihydroor otate hydrolysis, the bridging hydroxide attacks the re-face of dihydroorot ate with general base assistance by Asp 250. The carbonyl group is polarize d for nucleophilic attack by the bridging hydroxide through a direct intera ction with the beta -metal ion. During the cyclization of carbamoyl asparta te, Asp 250 initiates the reaction by abstracting a proton from N3 of the s ubstrate. The side chain carboxylate of carbamoyl aspartate is polarized th rough a direct electrostatic interaction with the binuclear metal center. T he ensuing tetrahedral intermediate collapses with C-O bond cleavage and ex pulsion of the hydroxide which then bridges the binuclear metal center.