Lipoic acid metabolism in Arabidopsis thaliana: cloning and characterization of a cDNA encoding lipoyltransferase.
Lipoic acid is an essential coenzyme required for activity of several key enzyme complexes, such as the pyruvate dehydrogenase complex, in the central metabolism. In these complexes, lipoic acid must be covalently attached to one of the component proteins for it to have biological activity. We report the cloning and characterization of Arabidopsis thaliana LIP2 cDNA for lipoyltransferase that catalyzes the transfer of the lipoyl group from lipoyl-acyl carrier protein to lipoate-dependent enzymes. This cDNA was shown to code for lipoyltransferase by its ability to complement an Escherichia coli lipB null mutant lacking lipoyltransferase activity. The expressed enzyme in the E. coli mutant efficiently complemented the activity of pyruvate dehydrogenase complex, but less efficiently than that of 2-oxoglutarate dehydrogenase complex. Comparison of the deduced amino acid sequence of LIP2 with those of E. coli and yeast lipoyltransferases showed a marked sequence similarity and the presence of a leader sequence presumably required for import into mitochondria. Southern and northern hybridization analyses suggest that LIP2 is a single-copy gene and is expressed as an mRNA of 860 nt in leaves. Western blot analysis with an antibody against lipoyltransferase demonstrated that a 29 kDa form of lipoyltransferase is located in the mitochondrial compartment of A. thaliana.
Wada
M
M
Department of Biology, Faculty of Sciences, Kyushu University, Ropponmatsu, Fukuoka, 810-8560 Japan. wadarcb@mbox.nc.kyushu-u.ac.jp
Yasuno
R
R
Jordan
S W
SW
Cronan
J E
JE
Wada
H
H
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D018744:DNA, Plant; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005816:Genetic Complementation Test; D007424:Intracellular Fluid / Q000378:metabolism; D008025:Ligases; D008074:Lipoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid; D008063:Thioctic Acid / Q000378:metabolism*
D001426:Bacterial Proteins; D018744:DNA, Plant; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; C490345:lplA protein, E coli; C089935:lplA protein, bacteria; D008063:Thioctic Acid; D000217:Acyltransferases; C438849:LIP2 lipoyltransferase, Arabidopsis; D008025:Ligases