Protein production, kinetic and biophysical characterization of three human dihydroorotate dehydrogenase mutants associated with Miller syndrome
Orozco Rodriguez, Juan Manuel LU ; Krupinska, Ewa LU ; Wacklin-Knecht, Hanna LU
and Knecht, Wolfgang
LU
(2022)
In Nucleosides, Nucleotides & Nucleic Acids
41(12).
p.1318-1336
- 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... (More)
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 .
(Less)
- author
- Orozco Rodriguez, Juan Manuel
LU
; Krupinska, Ewa
LU
; Wacklin-Knecht, Hanna
LU
and Knecht, Wolfgang
LU
- organization
- publishing date
- 2022-01-30
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Nucleosides, Nucleotides & Nucleic Acids
- volume
- 41
- issue
- 12
- pages
- 1318 - 1336
- publisher
- Taylor & Francis
- external identifiers
-
- pmid:35094635
- scopus:85124145260
- ISSN
- 1525-7770
- DOI
- 10.1080/15257770.2021.2023749
- language
- English
- LU publication?
- yes
- id
- 8fe29dda-ad59-4780-ad2f-21312990401c
- date added to LUP
- 2022-02-22 19:33:57
- date last changed
- 2024-12-13 07:09:11
@article{8fe29dda-ad59-4780-ad2f-21312990401c,
abstract = {{<p>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 .</p>}},
author = {{Orozco Rodriguez, Juan Manuel and Krupinska, Ewa and Wacklin-Knecht, Hanna and Knecht, Wolfgang}},
issn = {{1525-7770}},
language = {{eng}},
month = {{01}},
number = {{12}},
pages = {{1318--1336}},
publisher = {{Taylor & Francis}},
series = {{Nucleosides, Nucleotides & Nucleic Acids}},
title = {{Protein production, kinetic and biophysical characterization of three human dihydroorotate dehydrogenase mutants associated with Miller syndrome}},
url = {{http://dx.doi.org/10.1080/15257770.2021.2023749}},
doi = {{10.1080/15257770.2021.2023749}},
volume = {{41}},
year = {{2022}},
}