1j79
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Molecular Structure of Dihydroorotase: A Paradigm for Catalysis Through the Use of a Binuclear Metal Center
OverviewOverview
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 A resolution. Each subunit, of the homodimeric enzyme folds into a "TIM" barrel motif with eight, strands of parallel beta-sheet flanked on the outer surface by, alpha-helices. Unexpectedly, each subunit contains a binuclear zinc center, with the metal ions separated by approximately 3.6 A. Lys 102, which is, carboxylated, serves as a bridging ligand between the two cations. The, more buried or alpha-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 beta-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 A from the beta-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, beta-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.
About this StructureAbout this Structure
1J79 is a Single protein structure of sequence from Escherichia coli with ZN, ORO and NCD as ligands. Active as Dihydroorotase, with EC number 3.5.2.3 Full crystallographic information is available from OCA.
ReferenceReference
Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center., Thoden JB, Phillips GN Jr, Neal TM, Raushel FM, Holden HM, Biochemistry. 2001 Jun 19;40(24):6989-97. PMID:11401542
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