Peroxisomal plant 3-ketoacyl-CoA thiolase structure and activity are regulated by a sensitive redox switch

Abstract

The breakdown of fatty acids, performed by the beta-oxidation cycle, is crucial for plant germination and sustainability. beta-Oxidation involves four enzymatic reactions. The final step, in which a two-carbon unit is cleaved from the fatty acid, is performed by a 3-ketoacyl-CoA thiolase (KAT). The shortened fatty acid may then pass through the cycle again (until reaching acetoacetyl-CoA) or be directed to a different cellular function. Crystal structures of KAT from Arabidopsis thaliana and Helianthus annuus have been solved to 1.5 and 1.8 A resolution, respectively. Their dimeric structures are very similar and exhibit a typical thiolase-like fold; dimer formation and active site conformation appear in an open, active, reduced state. Using an interdisciplinary approach, we confirmed the potential of plant KATs to be regulated by the redox environment in the peroxisome within a physiological range. In addition, co-immunoprecipitation studies suggest an interaction between KAT and the multifunctional protein that is responsible for the preceding two steps in beta-oxidation, which would allow a route for substrate channeling. We suggest a model for this complex based on the bacterial system.

FIGURE 1.

The structure of KAT. A, schematic of AtKAT2 monomer: N-domain is shown in dark blue, C-domain in green, and L-domain in cyan. The secondary structure elements are labeled. B, superposition of KATs in open conformation. The r.m.s.d. values and sequence identities are given in supplemental Table S1. A detailed comparison of these structures is also included in the supplemental material (Discussion Fig. S1, and Table S1).

FIGURE 2.

Characterization of redox state dependent activity. A, AtKAT2 is inactive in the presence of CSSC and active in the presence of CSH. The rates of acetoacetyl-CoA cleaving by AtKAT2 were determined as a function of time and incubation with oxidant or reductant. 12.5 μm enzyme preparations were incubated at 25 °C in 35 mM Tris (pH 8.5), 0.1 m NaCl and 20 mm CSH (●), 10 mm CSSC (▾) or buffer (■). The effects of CSH (○) and CSSC (▿) on the substrate are included for reference. The incubations were sampled at the given time points by rapid mixing and 2500× dilution with 100 μm CoA, 50 μm acetoacetyl-CoA in a microcuvette with absorbance recorded at 233 nm. The final concentrations of AtKAT2, CSH, and CSSC in the reactions were 5, 8, and 4 nm, respectively. B, AtKAT2 changes tertiary structure upon incubation with CSSC. AtKAT2 was incubated with different ratios of [CSH]²/[CSSC] at 25 °C for 10 min, and tryptophan fluorescence was recorded at 333 nm with excitation wavelength 285 nm. The total concentration of CSH and CSSC was kept constant at 125 mg/liter. A two-state equation was fitted to the normalized (solid line) data and the midpoint of transition determined. C, Fluorescence spectra of reduced (solid line) and oxidized (dotted line) AtKAT2 at 1 and 0.03 m [CSH]²/[CSSC], respectively. Conditions as in B except emission recorded from 325 to 450 nm.

FIGURE 3.

Western blot analysis from co-immunoprecipitation studies. Lanes contained the following: lane 1, elution 1 from MFP beads; lane 2, load onto MFP beads; lane 3, peroxisomal lysate; lane 4, load onto KAT beads; lane 5, elution 3 from KAT beads; lane 6, elution 2 from KAT beads; lane 7, elution 1 from KAT beads; lane 8, molecular weight marker. A, Western blot developed with primary antibody against KAT. B, Western blot developed with primary antibody against MFP. A band is seen for MFP from the KAT2 pulldown suggesting a complex (black circle). Elution steps were performed only after extensive washing.

FIGURE 4.

Construction of the Arabidopsis KAT2·MFP2 complex model based on the prokaryotic multi-enzyme complex (PDB code 1WDK). A, Pseudomonas structure with MFP in medium gray and KAT in light gray. Region 1 (α-helical) and region 2 (β-hairpin 1) are highlighted in black. B, Arabidopsis model follows the same coloring scheme as A with AtMFP2 in medium gray and AtKAT2 dimer in light gray.

FIGURE 5.

Comparison of prokaryotic multi-enzyme complex with model complex of AtKAT2 and AtMFP2. A and C, Pseudomonas structure. B and D, Arabidopsis model. PfMFP and AtMFP are shown as an electrostatic surface colored according to electrostatic potential (from red (most electronegative) to white to blue (most electropositive)). PfKAT and AtKAT2 are shown as a rainbow-colored schematic according to conservation among prokaryotic and plant KATs, respectively (from blue (most conserved) to green to yellow to red (unconserved)).