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Human Molecular Genetics Advance Access published online on July 18, 2006
Human Molecular Genetics, doi:10.1093/hmg/ddl176
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© The Author 2006. Published by Oxford University Press. All rights reserved
Received April 27, 2006
Revised June 14, 2006
Accepted July 7, 2006

Article

The MELAS mutations 3946 and 3949 perturb the critical structure in a conserved loop of the ND1 subunit of mitochondrial Complex I

Marko Kervinen 1, Reetta Hinttala 2, Heli M. Helander 3, Sari Kurki 4, Johanna Uusimaa 3, Moshe Finel 5, Kari Majamaa 6, and Ilmo E. Hassinen 1 *

1 Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014 Oulu, Finland,
2 Clinical Research Center, Oulu University Hospital, FIN-90029 Oulu, Finland,; Department of Neurology, University of Oulu, FIN-90014 Oulu, Finland,
3 Department of Pediatrics, University of Oulu, FIN-90014 Oulu, Finland,
4 Department of Medical Chemistry, University of Helsinki, FIN-00014 Helsinki, Finland,
5 Department of Medical Chemistry, University of Helsinki, FIN-00014 Helsinki, Finland,; Drug Discovery and Development Technology Center, Faculty of Pharmacy, University of Helsinki, FIN-00014 Helsinki, Finland
6 Department of Neurology, University of Oulu, FIN-90014 Oulu, Finland,; Department of Neurology, University of Turku, FIN-20014 Turku, Finland.

* To whom correspondence should be addressed.
Ilmo E. Hassinen, E-mail: ilmo.hassinen{at}oulu.fi


  Abstract

The ND1 subunit gene of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a hot spot for mutations causing Leber hereditary optic neuropathy and several mutations causing the mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). We have used Escherichia coli and Paracoccus denitrificans as model systems to study the effect of mutations 3946 and 3949, which change conserved residues in ND1 and cause MELAS. The vicinity of these mutations was also explored with a series of mutations in charged residues. The 3946 mutation results in E214K substitution in human ND1. Replacement of the equivalent residue in E. coli with lysine or glutamine detracted from enzyme assembly and the assembled enzyme was inactive. However, the equivalent E234Q mutant enzyme in P. denitrificans failed to assemble completely (or was rapidly degraded). Also the corresponding substitution with aspartate decreased the enzyme activity in P. denitrificans and E. coli. The 3949-equivalent substitution, Y229H in E. coli, lowered the catalytic activity by 30 %. In addition, an activation of the enzyme during catalytic turnover was seen in this bacterial NDH-1, something that was even more pronounced in another mutant in the same loop, D213E. Several other mutations in this region decreased the enzyme activity. The studied MELAS mutations are situated in a matrix-side loop, which appears to be highly sensitive to structural perturbations. The results provide new information on the function of the region affected by the MELAS mutations 3946 and 3949 that is not obtainable from patient samples or current eukaryote models.


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