Expression, purification and crystallization of two peroxisomal acyl-CoA oxidases from<i>Arabidopsis thaliana</i>

Pedersen, Lise; Henriksen, A.

doi:10.1107/s0907444904007577

Cited by 7 publications

(10 citation statements)

References 25 publications

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“…In initial efforts to crystallize ACOX-1, we were only able to generate crystals of ACOX-1 without bound FAD, even when FAD was added to all buffers, as had been done for the purification and crystallization of certain plant ACOX enzymes (27). Serendipitously, we discovered that the ACOX-1(E434A) mutant can be readily crystallized with bound FAD.…”

Section: Resultsmentioning

confidence: 99%

Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans

Zhang

Jones

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Caenorhabditis elegans secretes ascarosides as pheromones to communicate with other worms and to coordinate the development and behavior of the population. Peroxisomal β-oxidation cycles shorten the side chains of ascaroside precursors to produce the short-chain ascaroside pheromones. Acyl-CoA oxidases, which catalyze the first step in these β-oxidation cycles, have different side chain-length specificities and enable C. elegans to regulate the production of specific ascaroside pheromones. Here, we determine the crystal structure of the acyl-CoA oxidase 1 (ACOX-1) homodimer and the ACOX-2 homodimer bound to its substrate. Our results provide a molecular basis for the substrate specificities of the acyl-CoA oxidases and reveal why some of these enzymes have a very broad substrate range, whereas others are quite specific. Our results also enable predictions to be made for the roles of uncharacterized acyl-CoA oxidases in C. elegans and in other nematode species. Remarkably, we show that most of the C. elegans acyl-CoA oxidases that participate in ascaroside biosynthesis contain a conserved ATP-binding pocket that lies at the dimer interface, and we identify key residues in this binding pocket. ATP binding induces a structural change that is associated with tighter binding of the FAD cofactor. Mutations that disrupt ATP binding reduce FAD binding and reduce enzyme activity. Thus, ATP may serve as a regulator of acyl-CoA oxidase activity, thereby directly linking ascaroside biosynthesis to ATP concentration and metabolic state.ascarosides | beta-oxidation | ATP | crystal structure | dauer pheromone

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Section: Resultsmentioning

confidence: 99%

Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans

Zhang

Jones

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Main chain regions 68 -87, 236 -242, 342-385, 537-621, and 692-695 have B-factors larger than 100 Å 2 . Pro 26 -Pro 27 and Ser 452 -Pro 453 are cispeptides (supplemental Fig. S4 online).…”

Section: Methodsmentioning

confidence: 99%

“…AtACX1 and AtACX4 were prepared as described by Pedersen and Henriksen (27). N-terminally His 6 -tagged AtACX3 was expressed from a pET24b vector construct prepared from the pda08625 clone (Riken) in RosettaGami2 (DE3) cells (Novagen).…”

Section: Methodsmentioning

confidence: 99%

The Multifunctional Protein in Peroxisomal β-Oxidation

Arent¹,

Christensen²,

Pye³

et al. 2010

Journal of Biological Chemistry

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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 ␤-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 acylbinding 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.Fatty acids are degraded by the sequential removal of two carbon units in a process known as ␤-oxidation (Fig. 1). This process is ubiquitous and takes its name from the oxidation taking place at the carbon atom ␤ to the carboxyl group. The discovery of a peroxisomal ␤-oxidation system was made in plants (1), and whereas peroxisomal ␤-oxidation in animals appears to be merely a fatty acid chain-shortening machine feeding mitochondrial ␤-oxidation, plant and fungal ␤-oxidation takes place almost entirely in the peroxisomes. The products of the reaction are H 2 O 2 , acetyl-CoA, reducing equivalents, and a variety of chain length-shortened acyl-containing molecules like the plant hormones jasmonic acid (2) and indole-3-acetic acid (3, 4).The conversion of storage triacylglycerols by ␤-oxidation provides metabolic energy and carbon skeletons for germination and early post-germinative seedling growth in oil seed plants (5) and metabolic energy and carbon for the production of hydrolytic enzymes in cereals (6). It is a salvage pathway for fatty acids during foliar senescence (7), supplies respiratory substrates to carbohydrate-deprived tissue (8), and at a lower level, is a constitutive property of all plant tissues most likely involved with membrane lipid turnover (7).Three proteins (each present as several isozymes) that host a total of four enzyme activities constitute the core of peroxisomal ␤-oxidation. Acyl-CoA oxidase (ACX) 6 oxidizes acyl-CoA to 2-trans-enoyl-CoA using FAD as co-enzyme. A multifunctional protein (MFP) adds water over the 2-trans-enoyl-CoA double bond and oxidizes the resultant L-3-hydroxyacylCoA usin...

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“…The crystal form was non-isomorphous with the crystal form observed with the His-tagged enzyme (16). ACX4⅐AcAcCoA crystals belong to space group P3 2 21, a ϭ b ϭ 144.7 Å, c ϭ 149.2 Å, contain four molecules/asymmetric unit and have a Matthew's coefficient of 2.4 Å 3 /Da and a solvent content of 47%.…”

Section: Methodsmentioning

confidence: 89%

“…Cryoprotection was achieved by transfer of the crystals to 25% (v/v) glycerol by gradually increasing the concentration by 5%. Crystallization conditions for His-tagged ACX4 without AcAcCoA have been reported previously (16). The use of the non-His-tagged construct and inclusion of AcAcCoA in the crystallization conditions dramatically improved the reproducibility and quality of the ACX4 crystals.…”

Section: Methodsmentioning

confidence: 97%

Controlling Electron Transfer in Acyl-CoA Oxidases and Dehydrogenases

Mackenzie

Pedersen

Arent

et al. 2006

Journal of Biological Chemistry

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Plants produce a unique peroxisomal short chain-specific acyl-CoA oxidase (ACX4) for ␤-oxidation of lipids. The short chain-specific oxidase has little resemblance to other peroxisomal acyl-CoA oxidases but has an ϳ30% sequence identity to mitochondrial acyl-CoA dehydrogenases. Two biochemical features have been linked to structural properties by comparing the structures of short chain-specific Arabidopsis thaliana ACX4 with and without a substrate analogue bound in the active site to known acyl-CoA oxidases and dehydrogenase structures: (i) a solvent-accessible acyl binding pocket is not required for oxygen reactivity, and (ii) the oligomeric state plays a role in substrate pocket architecture but is not linked to oxygen reactivity. The structures indicate that the acyl-CoA oxidases may encapsulate the electrons for transfer to molecular oxygen by blocking the dehydrogenase substrate interaction site with structural extensions. A small binding pocket observed adjoining the flavin adenine dinucleotide N5 and C4a atoms could increase the number of productive encounters between flavin adenine dinucleotide and O 2 .The central pathway for fatty acid breakdown in higher plants is via peroxisomal ␤-oxidation. Fatty acids enter the cycle in the form of acyl-CoA thioesters, and during one round of ␤-oxidation, the acyl chain is shortened by a two-carbon unit and one acetyl-CoA molecule is produced. The first step in the peroxisomal ␤-oxidation cycle is the introduction of a double bond into the acyl-CoA substrate, resulting in the formation of 2-trans-enoyl-CoA. This reaction is a two-step reaction catalyzed by the family of acyl-CoA oxidases (ACXs) 2 requiring flavin adenine dinucleotide (FAD) as a cofactor. The cofactor gets reduced to FADH Ϫ in the first half-reaction concomitant with acyl-CoA oxidation. FADH Ϫ is reoxidized by molecular oxygen in the second step, thereby generating H 2 O 2 , an intracellular signaling molecule.Unlike plants, two parallel and distinct ␤-oxidation pathways exist in mammals, the peroxisomal ␤-oxidation pathway and a mitochondrial ␤-oxidation pathway. In mitochondrial ␤-oxidation, the first step is catalyzed by the acyl-CoA dehydrogenase family (ACD) (1), which is related to ACXs. The FADH Ϫ in ACDs is not, however, reoxidized by molecular oxygen but rather by another flavoprotein, the electron transfer flavoprotein, which transfers electrons to the electron transport chain. As a result, the oxidation of fatty acids/amino acids and the generation of ATP molecules are linked in mitochondrial ␤-oxidation (2). It is puzzling that natural selection has not forced plants to utilize the mitochondrial electron transport chain for general lipid oxidation despite the apparent ATP advantage of this pathway. This might reflect less stress on the ATP requirement and a higher demand for lipid turnover and excess oxygen management in plants.Six genes for ACX isozymes have been identified in Arabidopsis thaliana. Five encode for proteins of ϳ75 kDa (3-5). The proteins have different but overlapping s...

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Expression, purification and crystallization of two peroxisomal acyl-CoA oxidases fromArabidopsis thaliana

Cited by 7 publications

References 25 publications

Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans

Structural characterization of acyl-CoA oxidases reveals a direct link between pheromone biosynthesis and metabolic state in Caenorhabditis elegans

The Multifunctional Protein in Peroxisomal β-Oxidation

Controlling Electron Transfer in Acyl-CoA Oxidases and Dehydrogenases

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