PDBsum entry: 2z27

Hydrolase

PDB-id: 2z27

Contents

Description

Header details

Header records

References

PROCHECK

Protein chains

343 a.a. *

Ligands

DOR

NCD

Metal ions

_ZN ×4

Waters ×590

* Residue conservation analysis

Tools

Clefts Calculation

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.

UniProt:

Chains A, B: P05020 (PYRC_ECOLI)

Seq:

Struc:

Seq:

348 a.a.

Struc:

343 a.a.*

Key:	PfamA domain
Secondary structure	CATH domain

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

Enzyme class:

E.C.3.5.2.3   [IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

Reaction:

(S)-dihydroorotate + H₂O = N-carbamoyl-L-aspartate (see diagram below)

Pathway:

Pyrimidine Biosynthesis

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

Related entries:

1xge
wild-type dihydroorotase from e. Coli
2e25
e. Coli dihydroortase t109s complexed with foa
2z24
2z25
2z26
2z28
2z29
2z2a
... plus others (see Header records)

Quick_links

Procheck

Clefts

Surface

Key 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.