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.

The Streptomyces coelicolor Lipoate-protein Ligase Is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains

“…Thus, the conserved Asp residue plays only a modest role in enzyme activity. This is consistent with the large number of interactions between the large and small subunits seen in the T. acidophilum ligase structure (18).…”

“…The mammalian protein functions only to transfer the lipoyl moiety from the adenylate to the lipoate protein (14). A third type of lipoate ligase is found in the thermophile archaeon Thermoplasma acidophilum, in which the ligase is composed of two separate proteins, LplA and LplB, encoded by adjacent genes (13,(15)(16)(17)(18). Both LplA and LplB are required for lipoyl-AMP formation, but LplA alone is sufficient for lipoyl transferase activity (13,18).…”

“…Both LplA and LplB are required for lipoyl-AMP formation, but LplA alone is sufficient for lipoyl transferase activity (13,18). A recent crystal structure shows that the two T. acidophilum proteins interact to form a structure with a domain orientation similar to that of the E. coli protein (18). Given the low sequence identity to known ligases plus conservation of the E. coli domain arrangement even in an enzyme in which the arrangement is not dictated by covalent bonding, the question arises as to the whether or not the S. coelicolor protein is a functional LplA protein able to catalyze the overall ligase reaction.…”

“…A third type of lipoate ligase is found in the thermophile archaeon Thermoplasma acidophilum, in which the ligase is composed of two separate proteins, LplA and LplB, encoded by adjacent genes (13,(15)(16)(17)(18). Both LplA and LplB are required for lipoyl-AMP formation, but LplA alone is sufficient for lipoyl transferase activity (13,18). A recent crystal structure shows that the two T. acidophilum proteins interact to form a structure with a domain orientation similar to that of the E. coli protein (18).…”

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

“…Indeed, a second protein had been proposed to interact with T. acidophilum LplA and thereby allow the complete reaction (55), and this proved to be the case (57,58). Hence, lipoate ligases can be bipartite, and a crystal structure of a bipartite enzyme has recently been reported (59). The lipoate ligase family has recently been shown to include a circularly permuted enzyme from the soil bacterium Streptomyces coelicolor.…”