Structure of Dihydroorotate Dehydrogenase B Paul Rowland, Sofie Nørager, Kaj Frank Jensen, Sine Larsen  Structure  Volume 8, Issue 12, Pages 1227-1238 (December 2000) DOI: 10.1016/S0969-2126(00)00530-X

Figure 1 Possible Pathway for the Transfer of Electrons from the Substrate Dihydroorotate (DHO) to the Electron Acceptor NAD+ The bold lines represent the flow of electrons in a reaction favored at higher pH values. However, the reaction is reversible, and at lower pH values the enzyme readily reduces orotate to dihydroorotate at the expense of [NADH + H+]. The transfer of electrons from DHO to FMN takes place in the PyrDB subunit; the PyrK subunit binds the [2Fe-2S] cluster and FAD. The latter cofactor is necessary for the ability to use NAD+ as an electron acceptor Structure 2000 8, 1227-1238DOI: (10.1016/S0969-2126(00)00530-X)

Figure 2 Structure of Dihydroorotate Dehydrogenase B (a) The overall structure of DHODB viewed along the crystallographic 2-fold axis. The secondary-structural elements are colored according to B factors. Residues with B factors below 20 Å2 are dark blue, and residues with B factors above 40 Å2 are bright red. The intersubunit salt bridges are shown as a ball-and-stick representation. The FMN and FAD groups are represented as stick models in chartreuse and pale turquoise, respectively. The [2Fe-2S] cluster is shown as brown (Fe) and yellow (S) spheres. (b) The PyrDB subunit color ramped, with the N terminus in blue and the C terminus in red. The labels of the secondary-structural elements correspond to those in Figure 3. (c) The PyrK subunit colored according to increasing numbers in the sequence, as in Figure 2b. The labels of the secondary-structural elements correspond to those in Figure 3 Structure 2000 8, 1227-1238DOI: (10.1016/S0969-2126(00)00530-X)

Figure 3 Sequences of the Two Subunits in DHODB The top shows the alignment of the sequences of PyrDA and PyrDB. The residues shown in magenta are conserved in both family 1 and family 2 of the dihydroorotate dehydrogenases; those marked in spring green are only conserved in family 1. The secondary-structural elements in PyrDA are shown in green, and those in PyrDB are in blue. The symbol α is used for both α helices and 310 helices. The bottom of the figure contains the sequence of the PyrK protein with the secondary-structural elements marked. The residues marked in red are also conserved in the NADPH ferredoxin reductase superfamily. The cysteine ligands of the iron-sulfur cluster are shown in yellow. This figure was prepared with ALSCRIPT [51] Structure 2000 8, 1227-1238DOI: (10.1016/S0969-2126(00)00530-X)

Figure 4 Comparison of the Subunits, PyrDB and PyrK, to Related Proteins (a) A superposition of the Cα traces of the PyrDB (blue) with the PyrDA subunit (red) from DHODA. The FMN groups are represented in green. (b) Cα trace of the PyrK subunit (blue) with the structure of PDR (magenta, apart from the [2Fe-2S] domain shown in yellow) superimposed. The [2Fe-2S] cluster and the FAD of PyrK are represented as green ball-and-stick models; the [2Fe-2S] cluster and the FMN group of PDR are colored black. (c) The Cα trace of the two subunits in the closely linked PyrDB-PyrK dimer is displayed in stereo. The Cα traces for both subunits are color ramped. The PyrDB subunit is at the top with colors going from red to blue and the numbers of the residues given in black, and the PyrK subunit is at the bottom with colors from blue to violet and the residue numbers given in red Structure 2000 8, 1227-1238DOI: (10.1016/S0969-2126(00)00530-X)

Figure 5 The PyrDB-PyrK Interface (a) The part of the interface between the PyrDB and the PyrK subunits that contain the three redox centers in the uncomplexed DHODB structure. The residues from the PyrDB subunit are labeled with a red “A,” and those from the PyrK subunit are labeled with a green “B.” The violet broken line indicates the interface between the two subunits. (b) A superposition of the environment of the FMN and [2Fe-2S] clusters in the uncomplexed (yellow) and complexed (violet) structures. Residues from the two subunits are distinguished as in Figure 5a Structure 2000 8, 1227-1238DOI: (10.1016/S0969-2126(00)00530-X)

Figure 6 The Environment of the Two Flavin Groups and the Orotate Binding Site in DHODB (a) The environment of the FMN group in the uncomplexed PyrDB subunit with an open catalytic loop. Water molecules are shown as cyan spheres. (b) The DHODB-orotate complex structure showing the same view of the FMN group as in Figure 6a and containing a closed but slightly disordered catalytic loop. (c) A closeup view of the environment of the bound orotate in the complexed structure. (d) The environment of the FAD group in PyrK subunit in the uncomplexed structure. No significant changes were observed in the structure of the orotate complex Structure 2000 8, 1227-1238DOI: (10.1016/S0969-2126(00)00530-X)