Identification, Separation, and Characterization of Acyl-Coenzyme A Dehydrogenases Involved in Mitochondrial β-Oxidation in Higher Plants1

Abstract

The existence in higher plants of an additional ␤-oxidation system in mitochondria, besides the well-characterized peroxisomal system, is often considered controversial. Unequivocal demonstration of ␤-oxidation activity in mitochondria should rely on identification of the enzymes specific to mitochondrial ␤-oxidation. Acylcoenzyme A dehydrogenase (ACAD) (EC 1.3.99.2,3) activity was detected in purified mitochondria from maize (Zea mays L.) root tips and from embryonic axes of early-germinating sunflower (Helianthus annuus L.) seeds, using as the enzyme assay the reduction of 2,6-dichlorophenolindophenol, with phenazine methosulfate as the intermediate electron carrier. Subcellular fractionation showed that this ACAD activity was associated with mitochondrial fractions. Comparison of ACAD activity in mitochondria and acylcoenzyme A oxidase activity in peroxisomes showed differences of substrate specificities. Embryonic axes of sunflower seeds were used as starting material for the purification of ACADs. Two distinct ACADs, with medium-chain and long-chain substrate specificities, respectively, were separated by their chromatographic behavior, which was similar to that of mammalian ACADs. The characterization of these ACADs is discussed in relation to the identification of expressed sequenced tags corresponding to ACADs in cDNA sequence analysis projects and with the potential roles of mitochondrial ␤-oxidation in higher plants.

Acyl-CoA Dehydrogenases: Dynamic History of Protein Family Evolution

“…Experimental studies using respiratory-chain inhibitors to inhibit mitochondrial β -oxidation (Dieuaide et al 1993; Gerhardt et al 1995) made a strong argument for the existence of plant ACADs. Purified mitochondria from carbohydrate-starved maize root tips and sunflower embryos exhibited ACAD activity (Bode et al 1999); however, several of the putative plant ACADs based on sequence similarity with mammalian ACADs (Bode et al 1999) were identified later as peroxisomal acyl-CoA oxidases (De Bellis et al 2000; Hayashi et al 1999). The report of a single plant ACAD, putative IVD, identified from mitochondria of A. thaliana (Daschner et al 1999) was later followed by identification of an IVD from Solanum tuberosum (Faivre-Nitschke et al 2001).…”

The Promoter Activity of Isovaleryl-CoA Dehydrogenase-Encoding Gene (ivdA) fromAspergillus oryzaeIs Strictly Repressed by Glutamic Acid

“…To confirm that the cloned sequence encoded IVD, we measured the increased enzyme activity due to the putative ivdA gene under the control of a Taka-amylase gene promoter (PamyB). After submerged culture of the three transformants (PamyB-putative ivdA fusion gene introduced) at 30 C for 2 d using DPY-broth (2% dextin, 2% Polypepton, 1% yeast extract), the IVD activities in the cellfree extracts of the transformants were assayed by the method of Bode et al 2) All three transformants showed 2.5 to 5.0-fold increased IVD activities as compared to the host strain (A. oryzae HL-1036 (sC À ) originating in industrial strain HL-1034 3) ), so we concluded that the cloned sequence was the IVD-encoding gene ivdA (GenBank Accession no. AB177764).…”

Acyl-CoA dehydrogenase activity in pea cotyledon tissue during germination and initial growth

“…T h e major consensus that /?-oxidation is exclusively peroxisomal [ 13 has been challenged over recent years as increasing evidence in support of an additional, mitochondrial site has accrued, as reviewed by Masterson and Wood [2]. One [4]. Activity from pea cotyledon mitochondria was detected with a wide range of acyl-CoA substrates [5], with peaks at C,, C,, and C,, chain lengths indicating not one but a family of acyl-CoA dehydrogenases.…”