5.3. Structure of the unliganded yeast Δ3-Δ2-enoyl-CoA isomerase (III)
The structure of the yeast Δ3-Δ2-enoyl-CoA isomerase was solved by the MAD method using potassium perrhenate (KReO4) in determining the phases. The heavy atoms were incorporated into the hexagonal unliganded isomerase crystals by soaking the crystals in crystallization solution containing KReO4 prior to data collection at 100 K. The MAD data were 99.5 % complete with 5-fold redundancy. The resolution limit of the data was 2.15 Å. Perrhenate was found to be bound at two sites in the isomerase subunit, one of the sites being the active site (Fig. 11). The yeast enoyl-CoA isomerase structure complexed with perrhenate was refined to the R and free R-factors of 21.5 % and 25.7 %, respectively. Another unliganded data set without heavy atoms was collected at 2.5 Å to determine whether the bound perrhenate ion changes the conformation of the active site residues. The unliganded structure was solved by rigid body refinement (Murshudov et al. 1997) using the perrhenate-complexed structure as the model and refined to the R-factor of 20.1 % and the free R-factor of 25.1 %. The structures were found to be virtually identical, and they will therefore not be described individually.
The structure of the yeast enoyl-CoA isomerase consists of two domains, the N-terminal core domain and the C-terminal trimerization domain (see “Discussion”, Fig. 13A). The N-terminal domain contains the active site, and it is folded in the spiral-fold topology, each turn of the spiral being formed of two β -strands and an α-helix. The four turns of the core domain are followed by the trimerization domain, which consists of four α-helices, namely H7, H8 H9 and H10. The C-terminal domain folds over the core domain and covers the active site. A salt bridge between Lys233 in helix H8 and Asp135, which comes just after helix H3, anchors the domains together. This salt bridge is conserved in the known structures of hydratase/isomerase superfamily. There are three undefined regions in the structure of the yeast enoyl-CoA isomerase: the residues 1 to 7 in the N-terminus, the residues 271 to 280 in the C-terminus and the residues 74 to 88 near helix H2. These regions could not be built because of the lack of any features in the electron density map. Due to the disorder in the C-terminus, the PTS1 targeting sequence could not be detected, either. Other parts of the structure are well defined.
Three enoyl-CoA isomerase monomers, related by a crystallographic three-fold axis, form a disk-like trimer. The contacts between the subunits are extensive. The main contact region concerns the helix H8 of one subunit fitting into a complementary docking site shaped by residues from the helices H4, H5 and H9 of the adjacent subunit. In the crystal, two trimers are packed in such a way that they form hexamers with 32 crystallographic symmetry. The interaction between the trimers is, however, loose and there are only 30 intertrimer contacts.
In the unliganded structure, the mode of substrate binding of the yeast enoyl-CoA isomerase could be inferred by superimposing the ligand of the 4-CBA-CoA dehalogenase structure (Benning et al. 1996) onto the active site of the enoyl-CoA isomerase. The active site architecture is presented in Figure 11. Site-directed mutagenesis studies suggested that Glu158 would be the catalytic amino acid residue. From the ligand superposition, it could be seen that Glu158 is, indeed, situated so that it could exchange protons with the ligand molecule. In addition, there are no other protic
side chains in the immediate vicinity. The Glu158 side chain extends from a loop between the β -strand B4 and the helix H4. This region is part of the tight subunit-subunit interface. The side chain of Glu158 is hydrogen-bonded to only one protein atom, ND2 of Asn101. The thioester oxygen atom of the superimposed acyl-CoA points towards the NH groups of Ala70 and Leu126, which are thus suggested to form the conserved oxyanion hole (Fig. 11) (Holden et al. 2001). The catalytic site is accessible for acyl-CoA via an entrance lined by residues from the β -strands B2 and B4 as well as the C-terminal α-helix H10. Molecular-surface calculations with ICM (Abagyan & Totrov 1994) detected two large cavities near the active site. One of them is near Glu158 and is filled with water molecules (Fig. 11). The other cavity is mainly lined by apolar side chains, and no solvent or other molecules could be detected in this hydrophobic pocket. The apolar cavity is separated from the active site pocket by the side chain of Tyr38.