University of Oulu

Structure-function studies of the peroxisomal multifunctional enzyme type 2 (MFE-2)

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Author: Ylianttila, Mari1
Organizations: 1University of Oulu, Faculty of Science, Department of Biochemistry
Format: ebook
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 6.1 MB)
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Language: English
Published: 2005
Publish Date: 2005-11-29
Thesis type: Doctoral Dissertation
Defence Note: Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Kuusamonsali (Auditorium YB210), Linnanmaa, on December 9th, 2005, at 12 noon
Reviewer: Professor Wolfgang Schliebs
Professor Mauno Vihinen


Multifunctional enzyme type 2 (MFE-2) catalyses the second and the third reactions in the eukaryotic peroxisomal β-oxidation cycle, which degrades fatty acids by removing a two-carbon unit per each cycle. In addition to the 2-enoyl-CoA hydratase 2 and (3R)-hydroxyacyl-CoA dehydrogenase activities, mammalian MFE-2 has also a sterol carrier protein type 2-like (SCP-2L) domain. In contrast, yeast MFE-2 has two (3R)-hydroxyacyl-CoA dehydrogenases, one 2-enoyl-CoA hydratase 2 and no SCP-2L domain.

The physiological roles of yeast (3R)-hydroxyacyl-CoA dehydrogenases (A and B) were tested by inactivating them in turn by site-directed mutagenesis and testing the complementation of Saccharomyces cerevisiae fox-2 cells (devoid of endogenous MFE-2) with mutated variants of Sc MFE-2. Growth rates were lower for fox-2 cells expressing only a single functional domain than for those expressing the Sc MFE-2. Kinetic studies with purified Candida tropicalis MFE-2 and its mutated variants show that dehydrogenase A catalyzes the reaction more efficiently with the medium- and long-chain substrates than dehydrogenase B, which in turn is the only one active with the short chain fatty acids.

The structural basis of the substrate specificity difference of these two dehydrogenases was solved by X-ray crystallography together with docking studies. Protein engineering was used to produce a stabile, homogenous recombinant protein of C. tropicalis dehydrogenases in one polypeptide. The heterodimeric structure contains the typical fold of the short-chain alcohol dehydrogenase/reductase (SDR) family. Docking studies suggest that dehydrogenase A binds medium chain-length substrates as bended, whereas short chain substrates are dislocated, because they do not reach the hydrophobic contacts needed for anchoring the substrate to the active site, but are instead attracted by L44. Dehydrogenase B has a more shallow binding pocket and thus locates the short chain-length substrates correctly for catalysis. Thus the data provide clues for structural basis of the different substrate specificities.

The molecular basis of the patient mutations of MFE-2 (DBP deficiency) was studied using the recently solved crystal structures of rat (3R)-hydroxyacyl-CoA dehydrogenase, human 2-enoyl-CoA hydratase and SCP-2L. The predicted effect of the mutations on protein structure could in several cases be explained, and these data supported the conclusion that a genotype-phenotype correlation exists for DBP deficiency.

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Series: Acta Universitatis Ouluensis. A, Scientiae rerum naturalium
ISSN-E: 1796-220X
ISBN: 951-42-7896-8
ISBN Print: 951-42-7895-X
Issue: 448
Copyright information: © University of Oulu, 2005. This publication is copyrighted. You may download, display and print it for your own personal use. Commercial use is prohibited.