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 palmitoyltransferase I (CPT I)-like molecule from Drosophila melanogaster. The cDNA 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 adopts the same topology as its mammalian counterparts. The sequence contains all the residues that have been shown to be characteristic of carnitine acetyltransferases. Expression in the yeast Pichia pastoris confirmed that the cDNA does encode a CPT enzyme. The activity was found to be associated with a mitochondria-enriched fraction. Kinetic analysis revealed a K(m) for carnitine of 406 microM and a K(m) for palmitoyl-CoA of 105 microM. The CPT activity was very sensitive to inhibition by malonyl-CoA, with an IC(50) of 0.74 microM when the activity was assayed with 35 microM palmitoyl-CoA and 1% (w/v) albumin 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 equivalent residue (position 136) in Drosophila CPT I is arginine, implying that any basic residue might be compatible with such sensitivity. Evidence is presented that, unlike in mammals, Drosophila has only a single CPT I gene. Sequences 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.
Cellular Biochemistry Group, Hannah Research Institute, Ayr, KA6 5HL, U.K.
V N
VN
Jackson
J M
JM
Cameron
V A
VA
Zammit
N T
NT
Price
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002334:Carnitine O-Palmitoyltransferase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D012326:RNA Splicing; D051381:Rats; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase