PDBsum entry: 2z27

Hydrolase

PDB id: 2z27

Contents

Protein chains

343 a.a. *

Ligands

DOR

NCD

Metals

_ZN ×4

Waters ×590

* Residue conservation analysis

PDB id:

2z27

Name:

Hydrolase

Title:

Thr109ser dihydroorotase from e. Coli

Structure:

Dihydroorotase. Chain: a, b. Synonym: dhoase. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: pyrc. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.87Å R-factor: 0.148 R-free: 0.188

Authors:

M.Lee,M.J.Maher,J.M.Guss

Key ref:

M.Lee et al. (2007). Kinetic and Structural Analysis of Mutant Escherichia coli Dihydroorotases: A Flexible Loop Stabilizes the Transition State(,). Biochemistry, 46, 10538-10550. PubMed id: 17711307 DOI: 10.1021/bi701098e

Date:

17-May-07 Release date: 09-Oct-07

Protein chains

P05020  (PYRC_ECOLI) -  Dihydroorotase

Seq: 348 a.a.

Struc: 343 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.5.2.3 - Dihydroorotase.
• Pathway: Pyrimidine Biosynthesis
• Reaction: (S)-dihydroorotate + H₂O = N-carbamoyl-L-aspartate

(S)-dihydroorotate (Bound ligand (Het Group name = DOR) corresponds exactly) + H(2)O = N-carbamoyl-L-aspartate (Bound ligand (Het Group name = NCD) corresponds exactly)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: pyrimidine base biosynthetic process (3 terms)
• Biochemical function: hydrolase activity (5 terms)

Added reference

DOI no: 10.1021/bi701098e

Biochemistry 46:10538-10550 (2007)

PubMed id: 17711307

Kinetic and Structural Analysis of Mutant Escherichia coli Dihydroorotases: A Flexible Loop Stabilizes the Transition State(,).

M.Lee, M.J.Maher, R.I.Christopherson, J.M.Guss.

ABSTRACT

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-l-aspartate (CA-asp) to l-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. Two different conformations of the surface loop (residues 105-115) were found in the dimeric Escherichia coli DHOase crystallized in the presence of DHO (PDB code 1XGE). The loop asymmetry reflected that of the active site contents of the two subunits: the product, DHO, was bound in the active site of one subunit and the substrate, CA-asp, in the active site of the other. In the substrate- (CA-asp-) bound subunit, the surface loop reaches in toward the active site and makes hydrogen bonds with the bound CA-asp via two threonine residues (Thr109 and Thr110), whereas the loop forms part of the surface of the protein in the product- (DHO-) bound subunit. To investigate the relationship between the structural states of this loop and the catalytic mechanism of the enzyme, a series of mutant DHOases including deletion of the flexible loop were generated and characterized kinetically and structurally. Disruption of the hydrogen bonds between the surface loop and the substrate results in significant loss of catalytic activity. Furthermore, structures of these mutants with low catalytic activity have no interpretable electron density for parts of the flexible loop. The structure of the mutant (Delta107-116), in which the flexible loop is deleted, shows only small differences in positions of other substrate binding residues and in the binuclear zinc center compared with the native structure, yet the enzyme has negligible activity. The kinetic and structural analyses suggest that Thr109 and Thr110 in the flexible loop provide productive binding of substrate and stabilize the transition-state intermediate, thereby increasing catalytic activity.