PEROXISOMAL BETA-OXIDATION - PURIFICATION OF 4 NOVEL 3-HYDROXYACIL-COA DEHYDROGENASES FROM RAT-LIVER PEROXISOMES

Peroxisomes are capable of beta-oxidizing a variety of substrates incl uding the CoA esters of straight chain fatty acids, 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprosta nic acids. The first reaction of peroxisomal beta-oxidation is catalyz ed by an acyl-CoA oxidase. Rat liver peroxisomes contain three acyl-Co A oxidases: 1) palmitoyl-CoA oxidase, oxidizing straight chain acyl-Co As; 2) pristanoyl-CoA oxidase, oxidizing 2-methyl-branched acyl-CoAs; and 3) trihydroxycoprostanoyl-CoA oxidase, oxidizing the CoA esters of the bile acid intermediates (Van Veldhoven, P. P., Vanhove, G., Assel berghs, S., Eyssen, H. J., and Mannaerts, G. P. (1992) J. Biol. Chem. 267, 20065-20074). We have now investigated whether the third step of peroxisomal beta-oxidation, catalyzed by a 3-hydroxyacyl-CoA dehydroge nase, is also catalyzed by multiple enzymes, using the 3-hydroxyacyl-C oA derivatives of palmitic acid, 2-methylpalmitic acid, and trihydroxy coprostanic acid as the substrates to monitor the dehydrogenase activi ties. In order to avoid contamination with mitochondrial 3-hydroxyacyl -CoA dehydrogenases, highly purified peroxisomes from untreated rats w ere employed as the enzyme source. Subfractionation of the peroxisomes revealed that the major portion of the dehydrogenase activities with all three substrates was present in the peripheral membrane protein fr action. Separation of this fraction on various chromatographic columns resulted in the purification of the well known multifunctional protei n, a 78-kDa monomeric protein that displays 3-hydroxyacyl-CoA dehydrog enase plus hydratase activity, as well as of four additional novel deh ydrogenases with different substrate specificities. Three of the enzym es are monomeric proteins of 35 kDa, 56 kDa, and 79 kDa, respectively. The latter enzyme also displays hydratase activity. The fourth enzyme is a dimer of 89 kDa, the subunits of which form a doublet at 40 kDa. The exact physiological role of each of the 3-hydroxyacyl-CoA dehydro genases requires further investigation.