Sequencing and functional expression of the malonyl-CoA-sensitive carnitine palmitoyltransferase from Drosophila melanogaster

Using expressed sequence tag data, we obtained a cDNA for a carnitine palmi toyltransferase I (CPT I)-like molecule from Drosophila melanogaster. The c DNA encodes a 782-residue protein that shows 49% and 48% sequence identity with the rat liver and skeletal-muscle isoforms of CPT I respectively. The sequence has two predicted membrane-spanning regions, suggesting that it ad opts the same topology as its mammalian counterparts. The sequence contains all the residues that have been shown to be characteristic of carnitine ac etyltransferases. Expression in the yeast Pichia pastoris confirmed that th e cDNA does encode a CPT enzyme. The activity was found to be associated wi th a mitochondria-enriched fraction. Kinetic analysis revealed a K-m for ca rnitine of 406 mu M and a K-m for palmitoyl CoA of 105 mu M. The CPT activi ty was very sensitive to inhibition by malonyl-CoA, with an IC50 of 0.74 mu M when the activity was assayed with 35 mu M palmitoyl-CoA and 1% (w/v) al bumin at pH 7.0, A histidine residue at position 140 in rat liver CPT I has been indicated to be important for inhibition by malonyl-CoA. The equivale nt residue (position 136) in Drosophila CPT I is arginine, implying that an y basic residue might be compatible with such sensitivity. Evidence is pres ented that, unlike in mammals, Drosophila has only a single CPT I gene. Seq uences suggesting the existence of a splice variant in the 5' untranslated region were found; this was consistent with the existence of two promoters for the CPT I gene.