Gene isolation and characterization of two acyl CoA oxidases from soybean with broad substrate specificities and enhanced expression in the growing seedling axis

The first committed step in the -oxidation of fatty acids is catalyzed by the enzyme acyl-CoA oxidase (ACOX), which oxidizes a fatty acyl-CoA to a 2-trans-enoyl-CoA. To understand the role of -oxidation during seedling growth in soybean, two ACOX cDNAs were isolated by screening a seedling library with a DNA fragment obtained by RT-PCR by using degenerate oligonucleotides. The two cDNAs (ACX1;1 and ACX1;2) are 86% identical to each other at the nucleotide and the amino acid level. Their deduced amino acid sequences share significant homology with known acyl-CoA oxidases, including the conserved CGGHGY motif, a putative flavin mononucleotide binding site. In both sequences, the last three amino acids, ARL, represent a putative peroxisome targeting signal. The mRNA and protein of both cDNAs accumulated in all seedling tissues, with relatively stronger expression in the growing seedling axis and hypocotyl, and weaker expression in the cotyledon. Immunolocalization studies indicated that the two proteins were localized in the phloem cells of hypocotyl tissue. The two cDNAs were expressed in Escherichia coli and shown to possess acyl-CoA oxidase activity. With fatty acyl-CoA substrates of varying chain lengths, it was demonstrated that both ACX1;1 and ACX1;2 have broad substrate specificities (C₈–C₁₈). The stronger expression of ACX1;1 and ACX1;2 in the axis and hypocotyl tissue, the weaker expression in the oil-rich cotyledon tissue, and the broad substrate specificities suggest that the two acyl-CoA oxidases might play a general house-keeping role during soybean seedling growth, such as the turnover of membrane lipids.     

Lack of stereoselectivity of the peroxisome proliferation induced by 2-phenylpropionic acid: Evidence against a role for lipid disturbance in peroxisome proliferation

The significance of disturbances of lipid metabolism caused by xenobiotic acyl-CoAs as possible causes of peroxisomal proliferation has been studied with the enantiomers of 2-phenylpropionic acid (2-PPA), the (R)-enantiomer of which is converted to the acyl-CoA in rats while its (S)-antipode is not. rac-2-PPA (250 mg/kg/day ip × 3) was shown to be an hepatic peroxisomal proliferator in male Sprague–Dawley rats on the basis of increases in microsomal cytochrome P-450 content and lauric acid hydroxylation and hepatic CN^?-insensitive palmitoyl-CoA oxidation, a peroxisomal marker activity, while electron microscopy revealed a rise in the peroxisome/mitochondria ratio in hepatocytes. Further studies established the dose–response relationships for these biochemical changes. The (R)- and (S)-enantiomers were administered at a dose of 50 mg/kg/day ip × 3 and both were peroxisome proliferators of very similar potency. The effects of 100 mg/kg/day ip × 3 of the racemate, a dose giving ca. 75% of maximal response, were essentially additive of those of 50 mg/kg/day ip × 3 of its two component isomers. The stereoselectivity of acyl-CoA formation from the enantiomers of 2-PPA was confirmed by their differential inhibition of microsomal palmitoyl-CoA synthesis. Taken together, these data indicate that it is very unlikely that the acyl-CoA of 2-PPA plays any role in the peroxisomal proliferation which this compound causes in the rat. © 1994 Wiley-Liss, Inc.