Cloning and expression of a cDNA encoding bovine lipoyltransferase.
Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl-AMP to the specific lysine residue of the lipoate-dependent enzymes. We have isolated lipoyltransferase I (LipTI) and II (LipTII) from bovine liver (Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1994) J. Biol. Chem. 269, 16605-16609). N-terminal amino acid sequences of LipTI and LipTII were identical except that LipTI had an additional Asn residue on the N terminus. We cloned LipTII cDNA from a bovine liver cDNA library. The cDNA insert contained a 1119-base pair open reading frame encoding a precursor peptide of 373 amino acids including a mitochondrial targeting signal of 26 amino acids. The calculated molecular mass of the mature protein is 39,137 Da. The predicted amino acid sequence showed 35% identity with that of Escherichia coli lipoate-protein ligase A. Northern and Southern blot analyses showed a single band, and a single species of mRNA for lipoyltransferase was found by reverse transcription-polymerase chain reaction. Recombinant LipTII was expressed in E. coli and purified to apparent homogeneity. The Kmapp values of the recombinant enzyme for lipoyl-AMP and apoH-protein were comparable with those of native LipTII. An antibody raised against recombinant enzyme cross-reacted with LipTI and LipTII in a similar manner. The results suggest that LipTI and LipTII are derived from the same translated product but processed differently.
Fujiwara
K
K
Institute for Enzyme Research, the University of Tokushima, Tokushima 770, Japan.
Okamura-Ikeda
K
K
Motokawa
Y
Y
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011994:Recombinant Proteins; D000217:Acyltransferases; C109735:lipoyltransferase I; C109736:lipoyltransferase II