Δ³-Δ²-Enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae

Molecular and structural characterization

Anu Mursula

Department of Biochemistry, University of Oulu
Biocenter Oulu, University of Oulu

Abstract

The hydratase/isomerase superfamily consists of enzymes having a common evolutionary origin but acting in a wide variety of metabolic pathways. Many of the superfamily members take part in β-oxidation, one of the processes of fatty acid degradation. One of these β-oxidation enzymes is the Δ³-Δ ²-enoyl-CoA isomerase, which is required for the metabolism of unsaturated fatty acids. It catalyzes the shift of a double bond from the position C3 of the substrate to the C2 position.

In this study, the Δ ³-Δ ²-enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae was identified, overexpressed as a recombinant protein and characterized. Subsequently, its structure and function were studied by X-ray crystallography.

The yeast Δ ³-Δ ²-enoyl-CoA isomerase polypeptide contains 280 amino acid residues, which corresponds to a subunit size of 32 kDa. Six enoyl-CoA isomerase subunits assemble to form a homohexamer. According to structural studies, the hexameric assembly can be described as a dimer of trimers. The yeast Δ ³-Δ ²-enoyl-CoA isomerase is located in peroxisomes, the site of fungal β-oxidation, and is a necessary prerequisite for the β-oxidation of unsaturated fatty acids; the enoyl-CoA isomerase knock-out was unable to grow on such carbon sources.

In the crystal structure of the yeast Δ ³-Δ ²-enoyl-CoA isomerase, two domains can be recognized, the N-terminal spiral core domain for catalysis and the C-terminal α-helical trimerization domain. This overall fold resembles the other known structures in the hydratase/isomerase superfamily. Site-directed mutagenesis suggested that Glu158 could be involved in the enzymatic reaction. Structural studies confirmed this, as Glu158 is optimally positioned at the active site for interaction with the substrate molecule. The oxyanion hole stabilizing the transition state of the enzymatic reaction is formed by the main chain NH groups of Ala70 and Leu126.

The yeast Δ ³-Δ ²-enoyl-CoA isomerase hexamer forms by dimerization of two trimers, as in the other superfamily members. An extensive comparison of the five known structures of this family showed that the mode of assembly into hexamers is not a conserved feature of this superfamily, since the distance between the trimers and the orientation of the trimers with respect to each other varied. Marked differences were also detected between the two yeast enoyl-CoA isomerase crystal forms used in this study, one being crystallized at low pH and the other at neutral pH. The results suggest that the yeast Δ ³-Δ ²-enoyl-CoA isomerase could occur as a trimer at low pH.

Table of Contents

Acknowledgements

Abbreviations

List of original articles

1. Introduction

2. Review of the literature

2.1. Uptake and activation of fatty acids prior to β -oxidation

2.2. The β -oxidation cycle

2.2.1. β -Oxidation in mitochondria
2.2.2. β -Oxidation in peroxisomes
2.2.3. β -Oxidation in the yeast Saccharomyces cerevisiae

2.3. The requirement for auxiliary enzymes in β -oxidation

2.3.1. β -Oxidation of (poly)unsaturated fatty acids
2.3.2. β -Oxidation of α-methyl branched-chain fatty acids

2.4. Hydratase/isomerase superfamily of enzymes

2.4.1. Members of the superfamily
2.4.2. Structure and function of the hydratase/isomerase proteins

3. Aims of the present study

4. Materials and methods

4.1. Strains and plasmids

4.2. Saccharomyces genome database

4.3. Gene disruption (I)

4.4. Analysis of transcriptional regulation (I)

4.5. Subcellular localization (I)

4.6. Construction of the ECI1 expression vector (I)

4.7. Expression of the recombinant protein (I)

4.8. Protein purification (I, II)

4.9. Enzyme assays (I)

4.10. Site-directed mutagenesis (III)

4.11. Determination of native molecular mass (I, III, IV)

4.12. Crystallization (II, III, IV)

4.13. X-ray diffraction data collection and processing (II-IV)

4.13.1. Data from unliganded crystals (II)
4.13.2. Multiwavelength anomalous dispersion (MAD) data (III)
4.13.3. Data from octanoyl-CoA complexed crystals (IV)

4.14. Structure determination by the MAD method and refinement of the structure (III)

4.15. Structure determination by the molecular replacement method and refinement of the structure (IV)

4.16. Structure analysis and validation (III-IV)

4.17. Comparison of the structures belonging to the hydratase/isomerase superfamily (IV)

5.1. Characterization of ECI1 and its gene product, Eci1p (I)
5.2. Crystallization of yeast Δ³-Δ²-enoyl-CoA isomerase (II, IV)
5.3. Structure of the unliganded yeast Δ³-Δ²-enoyl-CoA isomerase (III)
5.4. Structure of Δ³-Δ²-enoyl-CoA isomerase complexed with octanoyl–CoA (IV)

6. Discussion

6.1. ECI1 encodes for a monofunctional peroxisomal Δ^3–Δ^2–enoyl–CoA isomerase (I)

6.2. The structures of the unliganded and the octanoyl-CoA-complexed yeast Δ³-Δ²-enoyl-CoA isomerase and the differences between them (III, IV)

6.3. Comparison of the yeast Δ³-Δ²-enoyl-CoA isomerase structures with the other structures within the hydratase/isomerase superfamily (III, IV)

6.3.1. Comparison at the monomer level
6.3.2. Comparison at the hexamer level

7. Conclusions

8. Future perspectives

List of Tables
1. The hydratase/isomerase superfamily members with known sequence and function.
2. The kinetic parameters of Δ³-Δ²-enoyl-CoA isomerases.

List of Figures
1. Activation of long-chain fatty acids and their transport into mitochondria for β -oxidation (modified from Eaton et al. 1996, Kerner & Hoppel 2000). The long-chain acyl-CoA synthetase, which activates long-chain fatty acids to long-chain acyl-CoAs in an ATP-dependent manner, is located on the outer mitochondrial membrane (OMM). The carnitine palmitoyltransferase I (CPT I), acylcarnitine carnitine translocase (translocase) and CPT II couple the acyl moiety to carnitine, shuttle the acylcarnitine through the inner mitochondrial membrane (IMM) and regenerate acyl-CoA, respectively.
2. The β -oxidation cycle. The intermediates of the pathway are shown. The four reactions are catalysed by 1. acyl-CoA dehydrogenase in mitochondria or acyl-CoA oxidase in peroxisomes, 2. 2-enoyl-CoA hydratase, 3. 3-hydroxyacyl-CoA dehydrogenase, and 4. 3-ketoacyl-CoA thiolase. The acyl-CoA shortened by two carbon atoms can enter subsequent cycles of β -oxidation.
3. The routes for the degradation of double bonds in unsaturated fatty acids. The dashed arrows indicate reactions of the classical β -oxidation cycle and the solid arrows reactions catalysed by auxiliary enzymes; DECR, 2,4-dienoyl-CoA reductase; ECI, Δ³-Δ²-enoyl-CoA isomerase; DECI, Δ^3,5-Δ^2,4-dienoyl-CoA isomerase. The even-numbered double bonds are degraded via the reductase-dependent route (A). The odd-numbered double bonds can be oxidized via either the isomerase-dependent pathway (B) or the route that also requires dienoyl-CoA isomerase and dienoyl-CoA reductase (C).
4. α-Methylacyl-CoA racemase is required for the β -oxidation of pristanoyl-CoA, which is the α–oxidation product of phytanoyl-CoA, and for bile acid synthesis. The peroxisomal β -oxidation steps include the action of branched-chain acyl-CoA oxidase, MFE-2 and SCPx. The figure was modified from Ferdinandusse et al. 2000.
5. The fold of the rat 2-enoyl-CoA hydratase-1 (pdb-entry code 1DUB) monomer. The N- and C-termini are marked, as are also the α-helices H1-H10. The β -strands are shown as arrows. The ligand acetoacetyl-CoA bound at the active site is presented as a ball-and-stick model.
6. The reaction mechanisms of rat 2-enoyl-CoA hydratase-1 (A) and Δ³-Δ²-enoyl-CoA isomerase (B) (adopted from Kiema et al. 1999). The reversible reaction of 2-enoyl-CoA hydratase-1 is shown in the direction of dehydration. In the reverse reaction, the hydration, Glu144, the general base activates a water molecule for an attack at the C3 of the trans-2-enoyl-CoA. The oxyanion hole residues Ala98 and Gly141 stabilize the negative charge forming on the thioester carbonyl oxygen during the intermediate state of the reaction. Glu164 acts as a catalytic acid and protonates C2 to form L-3-hydroxyacyl-CoA. In the reaction mechanism of enoyl-CoA isomerase, a double bond is shifted from C3 to C2. The catalytic base B1, in the case of the rat mitochondrial enoyl-CoA isomerase Glu165, abstracts a proton from C2, leading to an anionic transition state. Another unknown residue (B2) donates a proton to the C4, completing the reaction. The intermediates of the reactions are shown in brackets.
7. The dehalogenation of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA by the 4-CBA-CoA dehalogenase from Pseudomonas (modified from Benning et al. 1996 and Taylor et al. 1997). The reaction involves three intermediates: 1. The Meisenheimer complex, where Asp145 attacks the C4 of the benzoyl ring. The negative charge on the thioester carbonyl oxygen is stabilized at the oxyanion hole formed by Phe64 and Gly114. 2. Chloride leaves the complex and an arylated enzyme is generated. 3. His90 activates a water molecule for attack, and a tetrahedral intermediate is formed. The product 4-HBA-CoA leaves this complex and the catalytic residues are regenerated. The charged intermediates are shown in brackets.
8. The reaction catalyzed by Δ^3,5-Δ^2,4-dienoyl-CoA isomerase (modified from Modis et al. 1998). Glu196 acts as a catalytic base in the rat enzyme and abstracts a proton from the C2 of the substrate, 3,5-dienoyl-CoA. The acid Asp204 donates the proton to C6, and the product 2,4-dienoyl-CoA is released. The transition state of the reaction can be inferred from the Δ^3–Δ²-enoyl-CoA isomerase reaction in Fig. 6.
9. The decarboxylation of methylmalonyl-CoA to propionyl-CoA catalyzed by the methylmalonyl-CoA decarboxylase from E. coli (adapted form Benning et al. 2000). Tyr140 orients the carboxylate group in such a way that the decarboxylation process is facilitated. The anionic intermediate is stabilized by hydrogen bonding to His66 and Gly110. It is unclear which residue (A) serves as a catalytic acid and protonates the intermediate so that propionyl-CoA can form.
10. Yeast Δ³-Δ²-enoyl-CoA isomerase crystal forms. (A) A tetragonal unliganded crystal. The longest dimension of the crystal is 0.3 mm. (B) A hexagonal crystal belonging to the space group P6₃22. This crystal form was grown at pH 5.5 and was used in the determination of the perrhenate-complexed and the unliganded yeast isomerase structures. The longest dimension of the crystal is 0.3 mm. (C) The octanoyl-CoA-complexed crystal form crystallized at pH 7.0. The longest dimension of the crystals is 0.15 mm.
11. The hydrogen-bonding network at the active site of the yeast enoyl-CoA isomerase. The distances between the hydrogen-bonded atoms are shown. A perrhenate ion is bound at the active site contacting the catalytic residue Glu158. The bindings of W390 and the ReO4^- ion are mutually exclusive, as they compete for the same binding site. The water molecules W365, W302, W23, W35, W19, W423, W15 are part of the buried water cluster. W126 is in contact with the bulk solvent. W390 is in the oxyanion hole formed by N(Ala70) and N(Leu126). In the octanoyl-CoA-complexed enoyl-CoA isomerase structure, W390 is replaced by the thioester oxygen atom of the ligand. Asn248 and Glu251 extend from the helix H9 of the C-terminal domain, and the residues Ile156, Thr157 and the catalytic Glu158 are located just before the helix H4 (see original article III, Fig. 2). Tyr38 leads to the empty apolar cavity and is only weakly hydrogen-bonded to Leu126. In the octanoyl-CoA complex, the position of Tyr38 has not changed and the empty apolar cavity behind it still exists.
12. Octanoyl-CoA bound at the active site of the yeast Δ³-Δ²-enoyl-CoA isomerase. The catalytic residue Glu158 as well as the residues forming the oxyanion hole, Ala70 and Leu126, are shown. The main chain NH groups of Ala70 and Leu126 are hydrogen-bonded to the thioester oxygen of the octanoyl-CoA molecule. The electron density map drawn around the ligand was calculated after an omit refinement. For this, the atoms of octanoyl-CoA were omitted from the model and 10 cycles of refinement with REFMAC (Murshudov et al. 1997) were performed, after which a difference electron density map was calculated. The map is contoured at 2σ.
13. The overall fold of the low pH form (A) and the neutral pH form (B) of the yeast Δ^3–Δ²-enoyl-CoA isomerase monomer. The most variable α-helix H2 and the C-terminal helices H7, H8, H9 and H10 are shown. In addition, in the neutral pH form structure (B) the new α-helix H2A and the bound octanoyl-CoA molecule are depicted.
14. The substrates of Δ³-Δ²-enoyl-CoA isomerase, trans- and cis-3-enoyl-CoA and the product, trans-2-enoyl-CoA. R is the remainder of the acyl chain.

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