Resumen: The lipoyl domains of the dihydrolipoyl acyltransferase (E2p, E2o) components of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes are specifically recognised by their cognate 2-oxo acid decarboxylase (E1p, E1o). A prominent surface loop links the first and second beta-strands in all lipoyl domains, close in space to the lipoyl-lysine beta-turn. This loop
was subjected to various modifications by directed mutagenesis of a sub-gene encoding a lipoyl domain of Escherichia coli E2p. Deletion of the loop (four residues) rendered the domain incapable of reductive acetylation by E. coli E1p in the presence of pyruvate, but insertion of a new loop (six residues) corresponding to that in the E2o lipoyl domain partly restored this ability, albeit with a much lower rate. However, the modified domain remained unable to undergo reductive succinylation by E1o in the presence of 2-oxoglutarate. Additional exchange of the two residues on the C-terminal side of the loop (V14A, E15T) had no effect. Insertion of a different four-residue loop also restored a limited ability to undergo reductive acetylation, but still significantly less than that of the wild-type domain. Exchanging the residue on the N-terminal side of the lipoyl-lysine beta-turn in the E2p and E2o domains (G39T), both singly and in conjunction with the loop exchange, had no effect on the ability of the E2p domain to be reductively acetylated but did confer a slight increase in susceptibility to reductive succinylation. All mutant E2p domains, apart from that with the loop deletion (LD), were readily lipoylated in vitro by E. coli lipoate protein ligase A; the E2p LD mutant could be lipoylated only at a significantly lower rate. Likewise, this domain exhibited 1D and 2D NMR spectra characteristic of a partially folded protein, whereas the spectra of mutants with modified loops were similar to those of the wild-type domain. The surface loop is evidently important to the structural integrity of the domain and may help to stabilize the thioester bond linking the acyl group to the reduced lipoyl-lysine swinging arm as part of the catalytic mechanism. Recognition of the lipoyl domain by its partner E1 appears to be a complex process and not attributable to any single determinant on the domain.