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CSA entry for 1j79
Original Entry
Title:
Hydrolase
Compound:
Dihydroorotase
Mutant:
No
UniProt/Swiss-Prot:
P05020-PYRC_ECOLI
EC Class:
3.5.2.3
Other CSA Entries:
Overview of all sites for 1j79
Homologues of 1j79
Entries for UniProt/Swiss-Prot: P05020
Entries for EC: 3.5.2.3
Other Databases:
PDB entry: 1j79
PDBsum entry: 1j79
UniProt/Swiss-Prot: P05020
IntEnz entry: 3.5.2.3
Literature Report:
Introduction:
Dihyrdoorotase catalyses the reversible interconversion of carbamoyl aspartate and dihydroorotate. This reaction is an essential part of the pathway for the biosynthesis of pyrimidine nucleotides. Sequence comparisons show that there are two general classes of dihydroorotases. Class II enzymes are all monofunctional proteins from Gram-negative bacteria and yeast. Class I enzymes, found in higher organisms, are much larger and typically contain several enzyme activities as exemplified by CAD, a multifunctional enzyme found in mammals, insects and moulds. This protein combines the first three enzymes of the pyrimidine biosynthesis pathway: carbamoyl phosphate synthetase, aspartate carbamoylase, and dihydroorotase. Sequence homology within each class of enzymes is quite high (>40%) while that between the two classes is much lower. Both classes employ the same mechanism involving a binuclear zinc centre.
Mechanism:
Dihydroorotase uses a bi-nuclear zinc centre to catalyse the reversible formation and hydrolysis of dihydroorotate. In the hydrolysis reaction, the two zinc ions (alpha and beta) coordinate a bridging hydroxide ion that attacks the substrate carbonyl to form a tetrahedral intermediate. This process is facilitated by Asp 250 acting as a general base to deprotonate the attacking hydroxide, and by polarisation of the carbonyl group through coordination to the beta zinc ion. The tetrahedral intermediate and associated transition state is stabilised by coordination to both zinc ions. Collapse of this intermediate occurs with Asp 250 acting as a general acid to protate the departing amide nitrogen.
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Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASPA 250 251Sidechain
Acid/baseWater
Acid/baseSubstrate
Acts as a general base to deprotonate the attacking bridging hydroxide. Later acts as a general acid to protonate the amide nitrogen leaving group.
Evidence from paper Evidence concerns Evidence type
PubMed ID 15610022 Current protein Mutagenesis of residue
PubMed ID 11401542 Current protein Conservation of residue
PubMed ID 11401542 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ZNA 400 0
ElectrostaticTransition state
ElectrostaticWater
Activates the attacking water molecule by lowering its pKa to allow deprotonation. Coordinates and stabilises the tetrahedral intermediate/transition state.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11401542 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ZNA 401 0
ElectrostaticSubstrate
ElectrostaticTransition state
ElectrostaticWater
Polarises carbonyl of substrate for nucleophilic attack. Activates the attacking water molecule by lowering its pKa to allow deprotonation. Coordinates and stabilises the tetrahedral intermediate/transition state.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11401542 Current protein Residue is positioned appropriately (ligand position known)
References:
1
Mechanism of the dihydroorotase reaction
T. N. Porter and Y. Li and F. M. Raushel
Biochemistry. 43 (51) 16285-92
15610022
2
Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal centre
J. B. Thoden and G. N. Phillips and T. M. Neal and F. M. Raushel and H. M. Holden
Biochemistry 40 (24) 6989-97, (2001)
11401542
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