PDBsum entry 1jue

Oxidoreductase

PDB id: 1jue

Contents

Protein chains

311 a.a. *

Ligands

FMN ×2

ACY ×2

GOL ×5

Metals

_MG ×2

Waters ×607

* Residue conservation analysis

PDB id:

1jue

Name:

Oxidoreductase

Title:

1.8 a resolution structure of native lactococcus lactis dihydroorotate dehydrogenase a

Structure:

Dihydroorotate dehydrogenase a. Chain: a, b. Engineered: yes

Source:

Lactococcus lactis. Organism_taxid: 1358. Gene: pyrd. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

1.80Å R-factor: 0.180 R-free: 0.198

Authors:

S.Norager,S.Arent,O.Bjornberg,M.Ottosen,L.Lo Leggio,K.F.Jensen, S.Larsen

Key ref:

S.Nørager et al. (2003). Lactococcus lactis dihydroorotate dehydrogenase A mutants reveal important facets of the enzymatic function. J Biol Chem, 278, 28812-28822. PubMed id: 12732650 DOI: 10.1074/jbc.M303767200

Date:

24-Aug-01 Release date: 09-Sep-03

Protein chains

A2RJT9  (PYRDA_LACLM) -  Dihydroorotate dehydrogenase A (fumarate) from Lactococcus lactis subsp. cremoris (strain MG1363)

Seq: 311 a.a.

Struc: 311 a.a.

Enzyme reactions

• Enzyme class: E.C.1.3.98.1 - dihydroorotate oxidase (fumarate).
• Reaction: (S)-dihydroorotate + fumarate = orotate + succinate

(S)-dihydroorotate + fumarate (Bound ligand (Het Group name = ACY) matches with 50.00% similarity) = orotate + succinate

• Cofactor: FMN

FMN (Bound ligand (Het Group name = FMN) corresponds exactly)

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

reference

DOI no: 10.1074/jbc.M303767200

J Biol Chem 278:28812-28822 (2003)

PubMed id: 12732650

Lactococcus lactis dihydroorotate dehydrogenase A mutants reveal important facets of the enzymatic function.

S.Nørager, S.Arent, O.Björnberg, M.Ottosen, L.Lo Leggio, K.F.Jensen, S.Larsen.

ABSTRACT

Dihydroorotate dehydrogenases (DHODs) are flavoenzymes catalyzing the oxidation of (S)-dihydroorotate to orotate in the biosynthesis of UMP, the precursor of all other pyrimidine nucleotides. On the basis of sequence, DHODs can be divided into two classes, class 1, further divided in subclasses 1A and 1B, and class 2. This division corresponds to differences in cellular location and the nature of the electron acceptor. Herein we report a study of Lactococcus lactis DHODA, a representative of the class 1A enzymes. Based on the DHODA structure we selected seven residues that are highly conserved between both main classes of DHODs as well as three residues representing surface charges close to the active site for site-directed mutagenesis. The availability of both kinetic and structural data on the mutant enzymes allowed us to define the roles individual structural segments play in catalysis. We have also structurally proven the presence of an open active site loop in DHODA and obtained information about the interactions that control movements of loops around the active site. Furthermore, in one mutant structure we observed differences between the two monomers of the dimer, confirming an apparent asymmetry between the two substrate binding sites that was indicated by the kinetic results.

Selected figure(s)

Figure 1.
FIG. 1. Structures of the native and variant DHODAs. Top, the native DHODA dimer and the residues chosen for mutations. FMN (yellow) and orotate (orange) are shown as stick models. The N- and C-terminals of the two subunits of the dimer are indicated with an N and C, respectively. The catalytic active base Cys-130 and the mutated residues are illustrated as stick models and are color-coded according to their location in the sequence. Blue: Arg-50, Pro-56, Arg-57, and the cis-proline loop (42-58); pink: Ser-129, Cys-130, Pro-131, Lys-136, and the active site loop (129-138); violet: Asn-127 and the -strand 123-127; green: Asn-67 and the loop 67-75; turquoise: Asn-193 and the loop 191-195; and red: Lys-213 and the Lys-213-helix (211-214). Bottom, the A subunit of the native structure in the presence of DTT and absence of orotate and three mutant structures (K213E(Oro), P56A(Oro) and K136E) oriented as the dimer at the top and color-coded according to the temperature factors of the residues. The color code goes from blue to red with blue representing residues with B-factors below or equal to 5 Å2 and red corresponding to B-factors above or equal to 55 Å2.

Figure 4.
FIG. 4. Selected close-up views of native and mutant DHODAs. a, alignment of the subunit A of native DHODA orotate complex (violet) and the K213E(Oro) structure (green). FMN and orotate are shown as yellow and orange stick models, respectively. The figure visualizes the difference between the open active site loop and the closed active site loop. A different view of the active site highlighting the interactions between protein and orotate (in orange) is shown for the native orotate complex (b) and N67A(Oro) (c), with the FMN group colored in magenta. Hydrogen bonds are shown as black dotted lines and selected water molecules are represented as cyan spheres.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 28812-28822) copyright 2003.

Figures were selected by an automated process.