Abstract
The peroxisomal multifunctional enzyme type-1 (perMFE-1) is a monomeric
protein
of β-oxidation possessing 2-enoyl-CoA hydratase-1,
Δ3-Δ 2-enoyl-CoA
isomerase, and (3S)-hydroxyacyl-CoA dehydrogenase activities.
The amino-terminal part of perMFE-1 shows sequence similarity to mitochondrial
2-enoyl-CoA hydratases (ECH-1) and Δ3-Δ
2-enoyl-CoA isomerases, and belongs to the
hydratase/isomerase superfamily. Family members with known structures are either
homotrimers or homohexamers. The purpose of this work was to elucidate the
structure-function relationship of the rat perMFE-1 with special reference to the
hydratase/isomerase fold.
The structural adaptations required for binding of a long chain fatty acyl-CoA were
studied with rat ECH-1 via co-crystallization with octanoyl-CoA. The crystal
structure revealed that the long chain fatty acyl-CoA is bound in an extended
conformation. This is possible because, a flexible loop moves aside and opens a
tunnel, which traverses the subunit from the solvent space to the intertrimer
space.
Structural and enzymological studies have shown the importance of Glu144 and
Glu164 for the catalysis by ECH-1. In the present work the enzymological
properties of Glu144Ala and Glu164Ala variants of ECH-1 were studied. The
catalytic activity of hydration was reduced about 2000-fold. It was also
demonstrated that rat ECH-1 is capable of catalyzing isomerization. The
replacement of Glu164 with alanine reduced the isomerase activity 1000-fold,
confirming the role of Glu164 in both the hydratase and isomerase reactions. The
structural factors favoring the hydratase over the isomerase reaction were
addressed studying the enzymological properties of the Gln162Ala, Gln162Met, and
Gln162Leu variants. These mutants had similar enzymatic properties to wild type,
thus the catalytic function of the Glu164 side chain in the hydratase and
isomerase reaction does not depend on interaction with the Gln162 side chain.
The perMFE-1 was divided into five functional domains based on amino acid
sequence comparisons with the homologous proteins with known structures. Deletion
variants of perMFE-1 showed that the folding of an enzymatically active
amino-terminal hydratase/isomerase domain requires stabilizing interactions from
the two carboxy-terminal domains of perMFE-1. The last carboxy-terminal domain is
also required for the folding of the dehydrogenase part of perMFE-1. The
dehydrogenase part of perMFE-1 was crystallized.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-6577-7 |
| Date | 27 November 2001 |
| Creators | Kiema, T.-R. (Tiila-Riikka) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © University of Oulu, 2001 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3221, info:eu-repo/semantics/altIdentifier/eissn/1796-2234 |
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