Preparation of human dihydroorotate dehydrogenase for interaction studies with lipid bilayers

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition

“…A direct interaction between Q 10 and the enzyme is suggested by the increased retention upon rinsing that Hs Δ29DHODH displays on POPC/TOCL bilayers containing Q 10 in comparison to those without. This effect was both observed in our study here and in our previous study [ 43 ] using QCM-D. The location of ubiquinone at the center of the lipid bilayers and the lack of observable reorientation in the bilayer to bind DHODH are consistent with its large molecular size and branched chain structure, which make it poorly soluble in the phospholipids.…”

“…A total of 3 mL of protein solution was injected into the sample cell containing the lipid bilayer and the neutron reflectivity was measured after 30 min of incubation in the same solvent contrast, as well as after the sample cell was rinsed with buffer. Protein addition and rinsing were repeated in every contrast due to the protein interaction being partially reversible, as shown by our previous QCM-D measurements [ 43 ]. An overview of all of the bilayers and enzymes measured can be found in Supplementary Material Table S1 .…”

“…Our previous results, which were obtained with quartz crystal microbalance with dissipation monitoring (QCM-D) studies [ 43 ], showed that both Hs Δ29DHODH and Ec DHODH have very low binding to pure POPC lipid bilayers and bind reversibly, and that TOCL is a prerequisite for more stable binding. We therefore decided to investigate the structural basis of their interaction with the bilayers containing POPC and 10 mol% TOCL using NR.…”

“…Hs Δ29DHODH is comparable in enzymatic activity to the full-length Hs DHODH that is found naturally in mitochondria and the is most used variant of Hs DHODH studied throughout literature, also in interaction with lipids. Several reports of this, as well as comparisons of Class II DHODH with and without their N-terminal TM in vivo and in vitro studies have been published [ 13 , 35 , 40 , 41 , 42 , 43 , 44 ].…”

DHODH and cancer: promising prospects to be explored

“…Ubiquinone, utilizing the tunnel to approach the FMN cofactor for participating in the redox reaction, can easily diffuse into the mitochondrial inner membrane [ 18 , 19 ]. Ubiquinone Q 10 binds the signal peptide located in the N-terminus of DHODH, which covers for mitochondrial import and is a transmembrane domain with a microdomain interacting with the mitochondrial inner membrane [ 20 , 21 ] .…”