title: Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis.
abstract: 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-His(6)) 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.
authors.affiliation: Department of Biochemistry, Life Sciences Center, University of Missouri, Columbia, MO 65211, USA. jillcasteel@hotmail.com
authors.forename: Jill
authors.initials: J
authors.lastname: Casteel
authors.forename: Ján A
authors.initials: JA
authors.lastname: Miernyk
authors.forename: Jay J
authors.initials: JJ
authors.lastname: Thelen
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020134:Catalytic Domain; D060365:Chloroplast Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D019295:Computational Biology; D051047:Dihydrolipoyllysine-Residue Acetyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D054878:Lipoylation; D008239:Lysine / Q000378:metabolism; D056186:Metagenomics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D008063:Thioctic Acid / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D060365:Chloroplast Proteins; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D008063:Thioctic Acid; C586947:AT3G25860 protein, Arabidopsis; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D008239:Lysine