The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain.
The dihydrolipoamide S-acetyltransferase (E2) subunit of the maize mitochondrial pyruvate dehydrogenase complex (PDC) was postulated to contain a single lipoyl domain based upon molecular mass and N-terminal protein sequence (Thelen, J. J., Miernyk, J. A., and Randall, D. D. (1998) Plant Physiol. 116, 1443-1450). This sequence was used to identify a cDNA from a maize expressed sequence tag data base. The deduced amino acid sequence of the full-length cDNA was greater than 30% identical to other E2s and contained a single lipoyl domain. Mature maize E2 was expressed in Escherichia coli and purified to a specific activity of 191 units mg(-1). The purified recombinant protein had a native mass of approximately 2.7 MDa and assembled into a 29-nm pentagonal dodecahedron as visualized by electron microscopy. Immunoanalysis of mitochondrial proteins from various plants, using a monoclonal antibody against the maize E2, revealed 50-54-kDa cross-reacting polypeptides in all samples. A larger protein (76 kDa) was also recognized in an enriched pea mitochondrial PDC preparation, indicating two distinct E2s. The presence of a single lipoyl-domain E2 in Arabidopsis thaliana was confirmed by identifying a gene encoding a hypothetical protein with 62% amino acid identity to the maize homologue. These data suggest that all plant mitochondrial PDCs contain an E2 with a single lipoyl domain. Additionally, A. thaliana and other dicots possess a second E2, which contains two lipoyl domains and is only 33% identical at the amino acid level to the smaller isoform. The reason two distinct E2s exist in dicotyledon plants is uncertain, although the variability between these isoforms, particularly within the subunit-binding domain, suggests different roles in assembly and/or function of the plant mitochondrial PDC.
Thelen
J J
JJ
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.
Muszynski
M G
MG
David
N R
NR
Luethy
M H
MH
Elthon
T E
TE
Miernyk
J A
JA
Randall
D D
DD
D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D020224:Expressed Sequence Tags; D014644:Genetic Variation; D006801:Humans; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D003313:Zea mays / Q000201:enzymology*
D018076:DNA, Complementary; D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase