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Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis
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 in the primary sequence of the dihydrolipoyl S-acetyltransferase (E2) component. Arabidopsis thaliana plastidial E2 (AtplE2-1A-His6) was expressed in Escherichia coli. Analysis of recombinant protein by SDS-PAGE revealed a Mr 59,000 band. Supplementation of bacterial culture medium with l-lipoic acid (LA) 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 LA. Results from both intact and tryptic peptide mass determinations by MALDI–TOF MS confirmed that the mutant proteins were not lipoylated. The A. thaliana plastidial E2 subunit includes a single lipoyl-prosthetic group covalently attached to Lys96. Despite low primary sequence identity with bacterial E2, the plant E2 protein was recognized and modified by E. coli E2 lipoyl-addition system. Results from meta-genomic analysis suggest a β-turn is more important in defining the site for LA addition than a conserved sequence motif.
Miernyk, Jan A.
Thelen, Jay J.
amino acid sequences
polyacrylamide gel electrophoresis
Plant physiology and biochemistry 2011 Nov., v. 49, no. 11
Journal Articles, USDA Authors, Peer-Reviewed
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