Human Molecular Genetics Advance Access originally published online on December 1, 2004
Human Molecular Genetics 2005 14(2):327-334; doi:10.1093/hmg/ddi029
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Human Molecular Genetics, Vol. 14, No. 2 © Oxford University Press 2005; all rights reserved
Ferrochelatase consisting of wild-type and mutated subunits from patients with a dominant-inherited disease, erythropoietic protoporphyria, is an active but unstable dimer
1Department of Biotechnology, Kyoto Institute of Technology, Kyoto 606-8585, Japan and 2Department of Hygiene, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
* To whom correspondence should be addressed. Tel: +81 757247789; Fax: +81 757247760; Email: taketani{at}ipc.kit.ac.jp
Received September 3, 2004; Revised November 1, 2004; Accepted November 23, 2004
Erythropoietic protoporphyria (EPP) is an autosomal inherited disease of heme biosynthesis caused by a partial deficiency of the enzyme ferrochelatase. Patients with EPP show only 20–30% normal activity because of mutations in one of the alleles of the ferrochelatase gene. To clarify the molecular mechanisms of this low level of activity, we co-expressed human ferrochelatase carrying His- and HA-tags in a tandem fashion in Escherichia coli. Purification of the His-tag-containing enzyme revealed that the His-enzyme forms an oligomer in association with the HA-enzyme, and analysis by gel-filtration confirmed that the enzyme is a dimer (
80 kDa). Then we expressed homo- and heterodimers composed of the wild-type and engineered mutants of the enzyme (C395
, H157A, H263A, H388A) or mutants from EPP patients (I186T, M267I). The levels of homodimeric enzymes produced were low, and the activities of the purified homodimeric mutants were abolished. On the other hand, the heterodimers with wild-type and mutated subunits exhibited potential, but weak, activities without a marked change of Km values for substrates. These results showed that heterodimers containing normal and mutated subunits retain the enzymic activity, which is inconsistent with the hypothesis that ferrochelatase is only active when the dimer contains two normal subunits. Pretreatment at 42°C led to a rapid inactiviation of the heterodimeric mutants, indicating instability. Thus, we provide evidence that the instability of the heterodimer containing normal and mutated ferrochelatase as well as the low production levels due to the structural defect of the mutant protein, not the abolishment of the enzymic activity of the heterodimer, causes the weak activity in EPP patients.
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