Identification and cloning of two forms of liver peroxisomal fatty Acyl CoA Oxidase from the koala (Phascolarctos cinereus)

Abstract

In the present study, the cloning, expression and characterization of the rate-limiting enzyme of the peroxisomal beta-oxidation spiral, acyl CoA oxidase (AOX), from koala (Phascolarctos cinereus) liver is described. It has been previously reported that peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity was absent in koala liver [Comp. Biochem. Physiol. (C) 127 (2000) 327]. This activity is a measure of the overall peroxisomal beta-oxidation minus the final step catalysed by thiolase. Two 2039 bp koala liver AOX cDNAs, designated AOX1 and AOX2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala AOX cDNAs encode proteins of 662 amino acids. Transfection of the koala AOX cDNAs into Cos-7 cells resulted in the expression of proteins with palmitoyl-CoA oxidase activity. The apparent K(m) values for AOX1 and AOX2 cDNA-expressed enzymes were 28 and 38 microM, respectively, which are within the range of order of magnitude reported for rat and human purified AOX enzymes (approximately 10 microM). Northern analysis, utilizing the koala AOX1 cDNA as probe, detected a more intense AOX mRNA band in the koala liver as compared to rats and humans. Southern blot analysis of liver genomic DNA samples revealed a single AOX gene fragment of less than 14 kb in koalas, rat and humans, suggesting a single AOX gene. Collectively, the results of this study suggest that the absence of peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity in the koala liver is possibly due to deficiencies of one or more enzymes downstream of acyl-CoA oxidase and/or deficiencies of mitochondrial beta-oxidation enzymes.