1J79

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

Summary for 1J79

• Entry DOI: 10.2210/pdb1j79/pdb
• Descriptor: dihydroorotase, ZINC ION, OROTIC ACID, ... (5 entities in total)
• Functional Keywords: tim barrel, metalloenzyme, pyrimidine biosynthesis, hydrolase
• Biological source: Escherichia coli
• Total number of polymer chains: 2
• Total formula weight: 78420.33

Authors

Thoden, J.B.,Phillips Jr., G.N.,Neal, T.M.,Raushel, F.M.,Holden, H.M. (deposition date: 2001-05-16, release date: 2001-06-20, Last modification date: 2025-03-26)

Primary citation

Thoden, J.B.,Phillips Jr., G.N.,Neal, T.M.,Raushel, F.M.,Holden, H.M.
Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center.
Biochemistry, 40:6989-6997, 2001

PubMed Abstract: 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.

PubMed: 11401542
DOI: 10.1021/bi010682i

Experimental method

X-RAY DIFFRACTION (1.7 Å)