PDBsum entry 1jrb

Oxidoreductase

PDB id: 1jrb

Contents

Protein chains

311 a.a. *

Ligands

FMN ×2

ORO ×2

Waters ×358

* Residue conservation analysis

References listed in PDB file

Key reference

Title

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

Authors

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

Ref.

J Biol Chem, 2003, 278, 28812-28822. [DOI no: 10.1074/jbc.M303767200]

PubMed id

12732650

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.

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.

Secondary reference #1

Title

Active site of dihydroorotate dehydrogenase a from lactococcus lactis investigated by chemical modification and mutagenesis.

Authors

O.Björnberg, P.Rowland, S.Larsen, K.F.Jensen.

Ref.

Biochemistry, 1997, 36, 16197-16205. [DOI no: 10.1021/bi971628y]

PubMed id

9405053

Secondary reference #2

Title

The crystal structure of lactococcus lactis dihydroorotate dehydrogenase a complexed with the enzyme reaction product throws light on its enzymatic function.

Authors

P.Rowland, O.Björnberg, F.S.Nielsen, K.F.Jensen, S.Larsen.

Ref.

Protein Sci, 1998, 7, 1269-1279. [DOI no: 10.1002/pro.5560070601]

PubMed id

9655329

Figure 1.
Fig. 1. The reaction catalyzed y DHOD. The orotate and flavin atoms are numbered.

Figure 2.
Fig. 2. The of theomtatebinding site intheDHODAmonomer. moleculespresentinthenativeenzymeactivesie(WatersG, H,

The above figures are reproduced from the cited reference which is an Open Access publication published by the Protein Society

Secondary reference #3

Title

The crystal structure of the flavin containing enzyme dihydroorotate dehydrogenase a from lactococcus lactis.

Authors

P.Rowland, F.S.Nielsen, K.F.Jensen, S.Larsen.

Ref.

Structure, 1997, 5, 239-252. [DOI no: 10.1016/S0969-2126(97)00182-2]

PubMed id

9032071

Figure 4.
Figure 4. The DHODA dimer structure viewed from above the twofold axis. The orientation of subunit A is the same as that of Figure 3. Secondary structure elements are coloured according to B factor: residues with B factors below 20 Å2 are dark blue and residues with B factors above 40 Å2 are bright red. The three cavities inside the dimer are shown as probe occupied volumes, as calculated by the program VOIDOO [40] using a probe radius of 1.2 Å. The two cavities above the flavin molecules are coloured magenta, while the intersubunit void is shown in orange. The two water molecules inside the central void are shown as green spheres. The intersubunit Glu206-Lys296 salt bridges are shown in ball-and-stick representation. (Figure produced using the programs BOBSCRIPT and RASTER3D.)

The above figure is reproduced from the cited reference with permission from Cell Press

Secondary reference #4

Title

Purification and characterization of dihydroorotate dehydrogenase a from lactococcus lactis, Crystallization and preliminary X-Ray diffraction studies of the enzyme.

Authors

F.S.Nielsen, P.Rowland, S.Larsen, K.F.Jensen.

Ref.

Protein Sci, 1996, 5, 852-856. [DOI no: 10.1002/pro.5560050506]

PubMed id

8732756