Protein production, kinetic and biophysical characterization of three human dihydroorotate dehydrogenase mutants associated with Miller syndrome

Rodriguez, Juan Manuel Orozco; Krupinska, Ewa; Wacklin-Knecht, Hanna; Knecht, Wolfgang

doi:10.1080/15257770.2021.2023749

Cited by 1 publication

(1 citation statement)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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 ].…”

Section: Introductionmentioning

confidence: 99%

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

Rodriguez

Wacklin-Knecht

Clifton

et al. 2022

IJMS

Self Cite

View full text Add to dashboard Cite

The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH (HsΔ29DHODH) and the DHODH from Escherichia coli (EcDHODH) with ubiquinone (Q10) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q10. Q10 is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q10. We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q10 is mediated by enzyme penetration. We can also show that EcDHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than HsΔ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q10, as well as lipid composition, on enzyme binding.

show abstract