DHODH (dihydroorotate dehydrogenase) catalyzes a key step in the biosynthesis of pyrimidines, precursors of nucleic acids needed for DNA and RNA synthesis in all living organisms. Because of this, DHODH is a drug target for the inhibition of inflammation and investigated as a target to inhibit cell proliferation (cancer) and viral replication.  Human DHODH is a membrane-bound protein found in mitochondria that uses ubiquinone Q₁₀ as an electron acceptor. Using neutron reflectometry and lipid deuteration enabled us to determine the interaction mechanism between the enzyme’s catalytic domain and the ubiquinone embedded deep inside the lipid bilayer, as well as to compare the role of the enzyme’s N-terminal alpha-helical microdomain in human DHODH (HsΔ29DHODH) and a bacterial analogue from Escherichia coli (EcDHODH).

The benefit of using NR for this work was to inspect in situ the mechanism by which the enzyme binds to the lipid membranes and to detect the location of the Q₁₀ by using chain deuterated d₆₃-POPC lipids provided by ESS through the Deuteration and Macromolecular Xtallisation platform (DEMAX) pilot proposal call scheme launched first in 2019. We have shown that Q₁₀ is located at the centre of lipid bilayers and that both DHODH enzymes penetrate deeply into the outer membrane leaflet towards the Q₁₀. This interaction had an enzyme-dependent binding strength for different lipid bilayer compositions and the presence/absence of Q₁₀. This is the first time the membrane penetration has been observed for DHODH enzymes lacking a N-terminal transmembrane anchor.

Figure 1. Potential models for the interaction of Class II DHODHs with ubiquinone. Model 1: Q₁₀ bends up and reaches toward the enzyme at the outside of the lipid bilayer. Model 2: The enzyme penetrates into the outer lipid bilayer leaflet to reach the Q₁₀ at the center of the bilayer. Model 3: The enzyme partially penetrates the outer lipid bilayer leaflet and Q₁₀ bends up and reaches towards the enzyme. Model 2, marked by a green box, is the only scenario supported by the data. N-terminally truncated human DHODH (HsΔ29DHODH) or Escherichia coli DHODH (EcDHODH) are shown in blue and red, lipids in brown, Q₁₀ in purple and the solid support for the membrane in grey.

The research team from Lund University and ESS carried out time-of-flight neutron reflectometry on the Inter (ISIS) and D17 (ILL) reflectometers, with the inner mitochondrial membrane models formed in situ in the NR sample cell by vesicle fusion. The lipid structures before and after protein addition were solved by measuring four buffer contrasts consisting of different fractions of heavy water (D₂O, CM4 (66 vol% D₂O), CMSi (38 vol% D₂O), and H₂O).  Chain-deuterated d₆₃-POPC synthesized at the DEMAX platform at ESS was used to provide contrast to determine the location of ubiquinone Q10, as well as the model mitochondrial lipid TOCL (tetraoleyl cardiolipin). The interaction of the human and E.Coli DHODH was investigated as function of lipid composition in both mammalian and bacterial model membranes.  The presence of ubiquinone increases the binding of HsΔ29DHODH to bilayers consisting of synthetic lipids (POPC, TOCL), but the phospholipipid composition also has an effect on the binding of HsΔ29DHODH. A mammalian-like phospholipid mixture derived from yeast displays a significantly higher protein binding ability compared to synthetic lipids. Our also results indicate that the bacterial EcDHODH displays a higher degree of binding to POPC and TOCL compared to the truncated human enzyme (HsΔ29DHODH), and that its binding to model bacterial membranes containing also POPE and POPG, is higher compared to only POPC and TOCL.

We now aim to understand the effects of mutations found in human DHODH that cause a rare genetic disease, Miller Syndrome, on the enzyme’s biochemical activity and interaction with membranes. For more information see DOI: 10.1080/15257770.2021.2023749 & DOI: 10.1080/15257770.2022.2039393

This work was part of the doctoral thesis “Reconstitution of Membrane-Bound Enzymes for Neutron Scattering Studies: A Case Study of Human Dihydroorotate Dehydrogenase” by Juan Manuel Orozco Rodriguez (Orozco, M. (2022). Reconstitution of Membrane-Bound Enzymes for Neutron Scattering Studies: A Case Study of Human Dihydroorotate Dehydrogenase. Lund University (Media-Tryck)).This thesis was supported by Lund University, the European Spallation Source ERIC and the Institut Laue-Langevin. Staff of the Lund Protein Production Platform (LP3, www.lu.se/lp3) provided technical support for many of the experiments and the ISIS Neutron and Muon Source of the STFC Rutherford Appleton Laboratory, Didcot, UK and the Institut Laue-Langevin, Grenoble, France provided beamtime.

Financial support was provided by Lund University, the Royal Physiographic Society of Lund, the Erik Philip-Sörensen Foundation, and the Jörgen Lindström Foundation.

Cite: Orozco Rodriguez, J. M., Wacklin-Knecht, H. P., Clifton, L. A., Bogojevic, O., Leung, A., Fragneto, G. & Knecht, W. New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition. Int. J. Mol. Sci. 23, 2437 (2022).

For the original articles, please visit: https://www.mdpi.com/1422-0067/23/5/2437