Cloning and Expression of a cDNA Encoding Bovine Lipoyltransferase

• Published in: The Journal of biological chemistry Vol. 272; no. 51; pp. 31974 - 31978
• Main Authors: Fujiwara, Kazuko, Okamura-Ikeda, Kazuko, Motokawa, Yutaro
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.12.1997; American Society for Biochemistry and Molecular Biology
• Subjects: Acyltransferases - genetics; Acyltransferases - metabolism; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Blotting, Southern; Cattle; Cloning, Molecular; DNA, Complementary; Kinetics; Liver - enzymology; Molecular Sequence Data; Polymerase Chain Reaction; Recombinant Proteins - genetics; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.51.31974

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.