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

Abstract

Miller syndrome is a rare Mendelian disorder caused by mutations in the gene encoding human dihydroorotate dehydrogenase (DHODH). Human DHODH, a Class II DHODH, is an integral protein of the inner mitochondrial membrane (IMM) catalyzing the fourth step of the de novo pyrimidine biosynthesis pathway. Here we present a summary of the state of knowledge regarding Miller syndrome in the absence of any current review on the topic. We then describe the production and characterization of three distinct DHODH missense mutations (G19E, E52G, R135C) associated with Miller syndrome by means of enzyme kinetics and biophysical techniques. These human DHODH mutants were produced both in E. coli and in insect cells using the baculovirus expression vector system. We can show that the effects of these mutations differ from each other and the wild-type enzyme with respect to decreased enzymatic activity, decreased protein stability and probably disturbance of the correct import into the IMM. In addition, our results show that the N-terminus of human DHODH is not only a structural element necessary for correct mitochondrial import and location of DHODH on the outer side of the IMM, but also influences thermal stability, enzymatic activity and affects the kinetic parameters.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.2023749 .

• Authors: Juan Manuel Orozco Rodriguez, Ewa Krupinska, Hanna Wacklin-Knecht, Wolfgang Knecht
• Journal: Nucleosides, nucleotides & nucleic acids (Nucleosides Nucleotides Nucleic Acids) Vol. 41 Issue 12 Pg. 1318-1336 ( 2022) ISSN: 1532-2335 [Electronic] United States
• PMID: 35094635 (Publication Type: Review, Journal Article)

Chemical References

• Dihydroorotate Dehydrogenase
• Oxidoreductases Acting on CH-CH Group Donors

Topics

• Humans
• Dihydroorotate Dehydrogenase
• Oxidoreductases Acting on CH-CH Group Donors (genetics, metabolism)
• Escherichia coli (genetics)
• Abnormalities, Multiple (genetics)