| Δ3-Δ2-Enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae: Molecular and structural characterization | ||
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The Δ3-Δ2-enoyl-CoA isomerase (Eci1p) from S. cerevisiae, encoded by the ECI1 gene, is a monofunctional, peroxisomal enzyme catalyzing the isomerization of trans/cis-3-enoyl-CoA to trans-2-enoyl-CoA. Eci1p was shown to be, at neutral pH, a hexameric protein formed of six identical 32-kDa subunits. According to the structural studies, the subunits are assembled into a hexamer as a dimer of trimers.
The unliganded structure of yeast isomerase was determined using a crystal obtained at low pH, and the structure with an active site ligand from a crystal at neutral pH. The overall fold of the yeast Δ3-Δ2-enoyl-CoA isomerase was found to be typical for the hydratase/isomerase superfamily in that it had a N-terminal spiral core domain for catalysis and a C-terminal α-helical trimerization domain. Some conformational differences could, however, be detected between the two yeast isomerase crystal forms. These rearrangements correlated with the much tighter assembly of the isomerase trimers into hexamers at neutral pH compared to the loosely interacting trimers in the low pH form, suggesting that the assembly could be pH-dependent. A comparison with the other known structures belonging to the hydratase/isomerase superfamily further showed that the mode of assembly of the trimers into hexamers is not a conserved feature.
Site-directed mutagenesis and structural data indicated that Glu158 is the only catalytic residue of Eci1p involved in shuttling the proton from the C2 of the substrate to the C4 of the product. We suggest that the negatively charged transition state of the enzymatic reaction is stabilized by hydrogen bonding to the peptide NH groups of the conserved oxyanion hole residues, Ala70 and Leu126.