Characterization of the 2-enoyl thioester reductase of mitochondrial fatty acid synthesis type II in mammals
1University of Oulu, Faculty of Science, Department of Biochemistry
2University of Oulu, Biocenter Oulu
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|Academic dissertation to be presented, with the assent of the Faculty of Science of the University of Oulu, for public defence in Auditorium GO101, Linnanmaa, on December 4th, 2008, at 12 noon
Docent Peter Mattjus
Docent Sami Väisänen
A data base search using the amino acid sequence of Saccharomyces cerevisiae Etr1p, the last enzyme of mitochondrial fatty acid synthesis type II (FAS II), revealed a highly similar human protein, NRBF-1. Expression of NRBF-1 in a yeast etr1Δ strain rescued its respiratory deficiency. NRBF-1 resides in mitochondria in cultured HeLa cells. The recombinant NRBF-1 is enzymatically active, reducing 2E-enoyl-CoAs to acyl-CoAs in an NADPH-dependent manner. Altogether, our data showed that NRBF-1 is a mitochondrial 2-enoyl-CoA reductase/2-enoyl thioester reductase (MECR/ETR1), the human functional counterpart of yeast Etr1p. In addition, MECR was also isolated from bovine heart. It turns out that mammals contain a mitochondrial FAS II pathway, in addition to cytoplasmic FAS I.
To investigate the functional mechanism of MECR/ETR1 at the molecular level, the protein was crystallized and the crystal structure determined. The apo-structure of MECR/ETR1 contains two sulfates in the nucleotide binding site and the domain arrangement resembles the NADPH-containing holo-structure of yeast Etr1p. The predicted mode of NADPH-binding and kinetic data suggest that Tyr94 and Trp311 play critical roles in catalysis. A pocket was found in the structure extending away from the catalytic site that can accommodate fatty acyl chains up to 16 carbons. An acyl carrier protein (ACP) binding site was also suggested.
To study the physiological function of mouse Mecr, two lines of transgenic mice overexpressing Mecr were generated. The Mecr transgenic mice developed cardiac and mitochondrial abnormalities. The phenotyping was carried out using echocardiography, heart perfusion, histology, and endurance testing. Our results suggest Mecr plays a role in mitochondrial and heart function. Therefore, inappropriate expression of the genes of FAS II may result in the development of cardiomyopathy.
Acta Universitatis Ouluensis. A, Scientiae rerum naturalium
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