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ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Publications at this Location » Publication #267275
Title: Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis

Author
item CASTEEL, JILL - University Of Missouri
item Miernyk, Jan
item THELEN, JAY - University Of Missouri

Submitted to: Plant Physiology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/5/2011
Publication Date: 10/12/2011
Citation: Casteel, J., Miernyk, J.A., Thelen, J.J. 2011. Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis. Plant Physiology and Biochemistry. 49:1355-1361.

Interpretive Summary: The pyruvate dehydrogenase complex occupies a critical position in biochemistry, and is controlled by multiple non-overlapping mechanisms. The component enzymes of the complex are all chemically modified, and the modifications contribute to their activities. While the chemical modifications have been studied in bacteria, yeast, and animals, relatively little is known about them in plants. A molecular biology-based method to determine the position of one of the chemical modifications was developed. An independent method from analytical chemistry was used to verify the results. A mutant form of the protein was prepared that could not be chemically modified. The mutant protein was completely inactive, which demonstrated how important the modification is. This information will be useful to scientists in their efforts to improve agricultural crop production through both classical breeding and application of biotechnology strategies, in this case to modify soybean seed content.

Technical Abstract: Background: The catalytic enhancement achieved by the pyruvate dehydrogenase complex (PDC) results from a combination of substrate channeling plus active-site coupling. The mechanism for active-site coupling involves lipoic acid prosthetic groups covalently attached to Lys residues in the primary sequence of the dihydrolipoyl S-acetyltransferase (E2) component of the complexes. Depending upon the molecular architecture of the PDC, the E2 subunits have one to three lipoyl groups. Results: Arabidopsis thaliana plastidial E2 (AtplE2-1A-His6) was expressed in Escherichia coli with a His-tag purification aid. Analysis of the recombinant protein by SDS-PAGE revealed a Mr 59,000 band. Supplementation of the bacterial culture medium with 1 mM L-lipoic acid shifted the band to Mr 57,000. Intact mass determinations using matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) revealed the faster migrating E2 species was 189 Da larger than the slower migrating form, exactly the difference that would result from addition of a single lipoamide group. Results from systematic MALDI-TOF analysis of Lys-containing tryptic peptides derived from purified recombinant AtplE2-1A indicate that Lys96 is the site of lipoyl-addition. Analysis of Lys96 site-directed mutant proteins showed that they migrated as single species during SDS-PAGE when expressed in either the absence or presence of supplemental lipoic acid (LA). Results from both intact and tryptic peptide mass determinations by MALDI-TOF MS confirmed that the mutant proteins were not lipoylated. Conclusions: The A. thaliana plastidial E2 subunit includes a single lipoyl prosthetic group covalently attached to Lys96. Despite the low degree of overall E2 primary sequence identity, the plant E2 protein was recognized and modified by E. coli E2 lipoyl-addition system. Results from a meta-genomic analysis support the previous suggestion that a ß-turn is more important in defining the site for LA addition than a conserved primary sequence motif.