title: Post-translational modification in the archaea: structural characterization of multi-enzyme complex lipoylation.
abstract: Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, lipoylation is catalysed by LplA (lipoate protein ligase) or by LipA (lipoic acid synthetase) and LipB [lipoyl(octanoyl) transferase] combined. Whereas bacterial and eukaryotic LplAs comprise a single two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The T. acidophilum LplA-N structure is known, but the LplA-C structure is unknown and LplA-C's role in lipoylation is unclear. In the present study, we have determined the structures of the substrate-free LplA-N-LplA-C complex and E2lipD (dihydrolipoyl acyltransferase lipoyl domain) that is lipoylated by LplA-N-LplA-C, and carried out biochemical analyses of this archaeal lipoylation system. Our data reveal the following: (i) LplA-C is disordered but folds upon association with LplA-N; (ii) LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; (iii) the adenylate-binding region of LplA-N-LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; (iv) LplAN-LplA-C and E2lipD do not interact in the absence of substrate; (v) LplA-N-LplA-C undergoes a conformational change (the details of which are currently undetermined) during lipoylation; and (vi) LplA-N-LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
authors.forename: Mareike G
authors.initials: MG
authors.lastname: Posner
authors.forename: Abhishek
authors.initials: A
authors.lastname: Upadhyay
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Crennell
authors.forename: Andrew J A
authors.initials: AJ
authors.lastname: Watson
authors.forename: Steve
authors.initials: S
authors.lastname: Dorus
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Danson
authors.forename: Stefan
authors.initials: S
authors.lastname: Bagby
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D051047:Dihydrolipoyllysine-Residue Acetyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054878:Lipoylation; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013822:Thermoplasma / Q000201:enzymology*; D008063:Thioctic Acid / Q000737:chemistry / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D007527:Isoenzymes; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D008063:Thioctic Acid; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D010453:Peptide Synthases; C072322:lipoate-protein ligase