The multifunctional protein in peroxisomal beta-oxidation: structure and substrate specificity of the Arabidopsis thaliana protein MFP2

Abstract

Plant fatty acids can be completely degraded within the peroxisomes. Fatty acid degradation plays a role in several plant processes including plant hormone synthesis and seed germination. Two multifunctional peroxisomal isozymes, MFP2 and AIM1, both with 2-trans-enoyl-CoA hydratase and l-3-hydroxyacyl-CoA dehydrogenase activities, function in mouse ear cress (Arabidopsis thaliana) peroxisomal beta-oxidation, where fatty acids are degraded by the sequential removal of two carbon units. A deficiency in either of the two isozymes gives rise to a different phenotype; the biochemical and molecular background for these differences is not known. Structure determination of Arabidopsis MFP2 revealed that plant peroxisomal MFPs can be grouped into two families, as defined by a specific pattern of amino acid residues in the flexible loop of the acyl-binding pocket of the 2-trans-enoyl-CoA hydratase domain. This could explain the differences in substrate preferences and specific biological functions of the two isozymes. The in vitro substrate preference profiles illustrate that the Arabidopsis AIM1 hydratase has a preference for short chain acyl-CoAs compared with the Arabidopsis MFP2 hydratase. Remarkably, neither of the two was able to catabolize enoyl-CoA substrates longer than 14 carbon atoms efficiently, suggesting the existence of an uncharacterized long chain enoyl-CoA hydratase in Arabidopsis peroxisomes.

FIGURE 1.

β-Oxidation reaction cycle. ACX oxidizes acyl-CoA to 2-enoyl-CoA generating H₂O₂ via reduction of FAD. MFP adds H₂O over the newly formed double bond (ECH activity) and oxidizes the hydroxyl-acyl group to a ketoacyl (HACD activity). Finally, ketoacyl-CoA thiolase cleaves of a two-carbon unit in a reverse Claisen condensation reaction producing acetyl-CoA and leaving a shortened acyl-CoA ready for another chain-shortening reaction.

FIGURE 2.

The AtMFP2 ECH-domain. A, the ECH domain is yellow, the linker to the HACD domains orange, the N-terminal HACD domain green, and the C-terminal HACD domain light green. The position of the ECH active site is indicated by a sketch of the hydrogen bonds to the active site Glu amino acid residues (see inset). The red molecules in the sketch are hypothetical and based on the active site of monofunctional RnECH (59). B, solvent-accessible surface of the AtMFP2-ECH acyl-binding pocket rotated 180° around a horizontal axis. The 4-(N,N-dimethylamino)cinnamoyl-CoA substrate from a superposition of RnECH and the AtMFP-ECH domain is included. The N- and C-terminal parts of the flexible loop (Ser⁷¹–Tyr⁸⁸) are colored blue, and the cis-Pro²⁷ is cyan. C, mapping of consensus sequence to the ECH flexible loop region including all of the plant MFP sequences (top) from supplemental Fig. S2 online, the AtMFP2-like sequences (middle), and the AtAIM1-like sequences (bottom).

FIGURE 3.

The AtMFP2-HACD domain. A, overall structure of AtMFP2. The NAD⁺ co-factor (cyan) and an acetyl-CoA molecule (white) from the superimposed structure of PfMFP (Protein Data Bank code 1WDM) are included. B, close-up view of the HACD active site and the conserved active site residues (Ser⁴²⁸, His⁴⁴⁹, Glu⁴⁶¹, and Asn⁴⁹⁹). The 3-hydroxyacyl-CoA dehydrogenase signature is indicated by the magenta color of the backbone trace. The proposed reaction mechanism of AtMFP2 is based on the present crystal structure and the reaction mechanism and structures of human HACD NAD⁺ and its substrate analogue complexes (56, 70).

FIGURE 4.

Hydratase and dehydrogenase activity of recombinant AtMFP2 and AtAIM1. Neither AtMFP2 nor AtAIM1 efficiently degrade enoyl chains longer than C14-CoA. A, in situ 2-trans-enoyl-CoA substrate synthesis with a mixture of acyl-CoA oxidases were monitored at A₂₆₀. B, upon full conversion 0.5 nm MFP2 (black), AIM1 (gray), or buffer were added and the hydratase activity determined by recording A₂₆₀ decrease at 27 °C. After the initial determination, additional 5 nm MFP was added to the reactions to secure full conversion. C, after the reactions had run to completion, 1 mm NAD⁺ was added to each reaction, and the dehydrogenase activity was determined by recording the A₃₄₀ increase at 27 °C. D, after 1 h at 27 °C total production of NADH was determined by recording the A₃₄₀. No C16-CoA substrate was fed to this reaction from the hydratase reaction in B. Each reaction consisted of about 50 μm acyl-CoA substrate, AtACX1, AtACX3 and AtACX4 each at 150 nm, 175 mm Tris-HCl, pH 8.5, 2.5% (w/v) polyethylene glycol 400, and 40 pm catalase.

FIGURE 5.

Effect of Tween 20 on MFP activity and substrate profile. A, AtMFP2 dehydrogenase activity was assayed with increasing concentrations of Tween 20. B, the dehydrogenase activities of AtMFP2 (black) and AtAIM1 (gray) with C14-CoA and C16-CoA in the presence of 0.1% (w/v) Tween 20 were determined. The insert illustrates 2-trans-enoyl-CoA production of each substrate. Conditions in both assays were the same as in Fig. 4.

FIGURE 6.

BSA effect on MFP activity and substrate profile. A, AtMFP2 was titrated with BSA, and dehydrogenase activity was determined. B, substrate was prepared in the presence of 6 μm delipidated BSA, and hydratase activity was subsequently determined, C. D, dehydrogenase activity was determined with 150 times more MFP in the C16- and C18-CoA wells compared with 0.5 nm in the C14-CoA well. The data are not normalized to μm enzyme to show residual long chain dehydrogenase activity at high enzyme concentrations. Conditions otherwise are as described for Fig. 4.

FIGURE 7.

A slice of the solvent-exposed surface of the AtMFP2-HACD active site. The acetoacetyl-CoA and NAD⁺ included are superimposed molecules from the structure of PfMFP (13). An unoccupied pocket marked with a dotted line is observed extending from the 3-hydroxyl end of the substrate.