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 ␤-oxidation cycle, is crucial for plant germination and sustainability. ␤-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 Å 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 ␤-oxidation, which would allow a route for substrate channeling. We suggest a model for this complex based on the bacterial system.Fatty acids are fundamental biomolecules that are abundant in all life forms. With their enormous variation in chain length and degree of saturation, they are essential for energy storage, form structural entities in biomembranes, and serve as signaling molecules. Fatty acids are broken down in a cyclic manner, two carbons at a time, to generate a range of products by the process known as ␤-oxidation (1). In higher plants (2) and yeast (3) ␤-oxidation of all forms of fatty acid occurs in the peroxisomes. In plants, ␤-oxidation is essential for a plethora of physiological roles including responses to senescence and starvation, fatty acid turnover, and the regulation of plant lipid composition. Germinating seeds depend on ␤-oxidation for the mobilization and release of energy stored in the seed (4). Arabidopsis thaliana seeds deficient in ␤-oxidation enzymes are unable to germinate without an external sugar source; they have large and unusual peroxisomes and accumulate C16 -C20 fatty acids (5, 6). ␤-Oxidation is also responsible for the synthesis of jasmonic acid (7) and indole-3-acetic acid (auxin) via conversion from indole-3-butyric acid (8), which serve as crucial plant hormones regulating plant development and responses to biotic and abiotic stress. Hydrogen peroxide, produced as a byproduct during ␤-oxidation, is used by catalase to oxidize different toxins (e.g. alcohols) and plays an important role in cellular signaling (9).␤-Oxidation comprises four reactions. First, the CoA-activated acyl chain is oxidized to 2-trans-enoyl-CoA by an acylCoA oxidase (10), producing H 2 O 2 as a byproduct. The double bond is then reduced by 2-trans-enoyl hydratase forming L-3-hydroxyacyl-CoA followed by oxidation by NAD ϩ -dependent L-3-hydroxyacyl-CoA dehydrogenase. Both the hydratase and dehydrogenase activities are performed by a multifunction...

Coenzyme A-free activity, crystal structure, and rational engineering of a promiscuous β-ketoacyl thiolase from Ralstonia eutropha

“…The small pocket they create may explain why both of these biosynthetic thiolases are specific for acetyl-CoA as a priming acyl-CoA. Degradative thiolases, which often perform thiolysis on long β-ketoacyl substrates, do not contain the glutamine or α5 (e.g., PDBs 1AFW, 2WU9, and 3GOA, from the Saccharomyces cerevisiae peroxisome, the Arabidopsis thaliana peroxisome, and Salmonella typhimurium ) [26,27] (Figure 4c). The region where the glutamine and α5 would be located in degradative thiolases is hypothesized to bind fatty acyl chains.…”

The peroxisome: an update on mysteries

“…Indeed, peroxisomal enzymes have been recently shown to be sensible to oxidative switches modulating their enzymatic properties: catalase and malate synthase of castor beans were observed to be increasingly oxidized upon H 2 O 2 exposure; importantly, protein modification did not result in protein destruction, but increased the activity of the correspondent enzymes, thereby adapting them to elevated cellular ROS concentrations (Anand et al 2009). For ketoacyl-CoA thiolases from plants, a redox-sensitive switch between a reduced active and an oxidized inactivated protein conformation was reported, which could adjust peroxisomal FA b-oxidation rates to slow down the associated H 2 O 2 -production, when the cellular redox state was already high (Pye et al 2010). These examples illustrate how oxidative protein modifications can be used to regulate intracellular processes.…”

Redox-regulated Cargo Binding and Release by the Peroxisomal Targeting Signal Receptor, Pex5

“…Maintaining a reduced environment in the peroxisome lumen is crucial for Pex11-mediated peroxisome proliferation and the activity of peroxisomal 3-ketoacyl-CoA thiolase, an enzyme involved in fatty acid ␤-oxidation (31)(32)(33). Deletion of Saccharomyces cerevisiae glutathione peroxidase 1, an antioxidant using thioredoxin or glutathione for its reducing power, impaired peroxisome biogenesis (34).…”