The role of loop and beta-turn residues as structural and functional determinants for the lipoyl domain from the Escherichia coli 2-oxoglutarate dehydrogenase complex.

The lipoyl domain of the dihydrolipoyl succinyltransferase (E2o) component of the 2OGDH (2-oxoglutarate dehydrogenase) multienzyme complex houses the lipoic acid cofactor through covalent attachment to a specific lysine side chain residing at the tip of a beta-turn. Residues within the lipoyl-lysine beta-turn and a nearby prominent loop have been implicated as determinants of lipoyl domain structure and function. Protein engineering of the Escherichia coli E2o lipoyl domain (E2olip) revealed that removal of residues from the loop caused a major structural change in the protein, which rendered the domain incapable of reductive succinylation by 2-oxoglutarate decarboxylase (E1o) and reduced the lipoylation efficiency. Insertion of a new loop corresponding to that of the E. coli pyruvate dehydrogenase lipoyl domain (E2plip) restored lipoylation efficiency and the capacity to undergo reductive succinylation returned, albeit at a lower rate. Exchange of the E2olip loop sequence significantly improved the ability of the domain to be reductively acetylated by pyruvate decarboxylase (E1p), retaining approx. 10-fold more acetyl groups after 25 min than wild-type E2olip. Exchange of the beta-turn residue on the N-terminal side of the E2o lipoyl-lysine DK(A)/(V) motif to the equivalent residue in E2plip (T42G), both singly and in conjunction with the loop exchange, reduced the ability of the domain to be reductively succinylated, but led to an increased capacity to be reductively acetylated by the non-cognate E1p. The T42G mutation also slightly enhanced the lipoylation rate of the domain. The surface loop is important to the structural integrity of the protein and together with Thr42 plays an important role in specifying the interaction of the lipoyl domain with its partner E1o in the E. coli 2OGDH complex.