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Post-translational modification in the archaea: structural characterization of multi-enzyme complex lipoylation

DOI: 10.1042/BJ20121150 DOI Help

Authors: Mareike Posner (University of Bath) , Abhisehk Upadhyay (University of Bath) , Susan Crennell (University of Bath) , Andrew j. A. Watson (University of Bath) , Steve Dorus (University of Bath) , Michael Danson (Centre for Extremophile Research, University of Bath) , Stefan Bagby (University of Bath)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal , VOL 449 , PAGES 415 - 425

State: Published (Approved)
Published: January 2013

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.

Journal Keywords: Binding-Induced Folding; Lipoate Protein Ligase; Lipoyl Domain; Nmr Spectroscopy; Protein–Protein Interaction; X-Ray Crystallography

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I02-Macromolecular Crystallography

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