| Δ3-Δ2-Enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae: Molecular and structural characterization | ||
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Multiple amino acid sequence alignments of Ecip1 with other hydratase/isomerase superfamily members were performed by CLUSTALX (Thompson et al. 1997) to predict the catalytic residues of Eci1p (I, III, IV). The active site amino acid of the rat mitochondrial enoyl-CoA isomerase, Glu165 (Müller-Newen & Stoffel 1993), is not conserved in Eci1p, being replaced by a phenylalanine (Phe150). A protic side chain two residues before that, Tyr148, was suggested to be a possible candidate for a catalytic residue. Another option was Glu158, which corresponds to the active site residues of Δ3,5-Δ2,4-dienoyl-CoA isomerase (Modis et al. 1998) and 4-CBA-CoA dehalogenase (Benning et al. 1996, Yang et al. 1996), Asp204 and Asp145, respectively. Both Tyr148 and Glu158 were mutated to alanine by using the QuickChangeTM mutagenesis kit (Stratagene) following the manufacturer’s instructions. For the mutagenesis, oligonucleotides which contained the desired mutation were used in the PCR reaction: 5’–CAAGGTTTATTTGCTAGCGCCCTTTGCTAACTTAG-3’ and 5’–CTAAGTTAGCAAAGGGCGCTAGCAAATAAACCTTG-3’ for the Tyr148Ala mutation and 5’-CTTAGGACTAATTACCGCAGGTGGTACAACGGTC-3’ and 5’–GACCGTTGTACCACCTGCGGTAATTAGTCCTAAG-3’ for the Glu158Ala mutation. The ECI1 gene ligated into the pET3a-expression vector was used as the template and pfu as the DNA polymerase. The mutated proteins were expressed and purified as described, and their Δ3-Δ2-enoyl-CoA isomerase activity was measured.