Structural determinants of post-translational modification and catalytic specificity for the lipoyl domains of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

Abstract

The lipoyl domains of the dihydrolipoyl acyltransferase (E2p, E2o) components of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes are speci®cally recognised by their cognate 2-oxo acid decarboxylase (E1p, E1o). A prominent surface loop links the ®rst and second bstrands in all lipoyl domains, close in space to the lipoyl-lysine b-turn. This loop was subjected to various modi®cations 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 modi®ed 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 signi®cantly less than that of the wild-type domain. Exchanging the residue on the N-terminal side of the lipoyl-lysine b-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 signi®cantly lower rate. Likewise, this domain exhibited 1D and 2D NMR spectra characteristic of a partially folded protein, whereas the spectra of mutants with modi®ed 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.

Recognition of the lipoyl domain is the ultimate determinant of substrate channelling in the pyruvate dehydrogenase multienzyme complex

“…However, the E2p lipoyl domain is an excellent substrate (k cat /K m raised by a factor of 10 4 ). Moreover, the E2p and E2o lipoyl domains from E. coli (Graham et al, 1989;Jones et al, 2000b) function as substrates only for their cognate E1 s. Similar results have been reported for the E2p and E2o lipoyl domains of Azotobacter vinelandii (Berg et al, 1998). Thus, the lipoyl domain provides an elegant mechanism for substrate channelling such that reductive acylation is con®ned to a lipoyl group covalently attached to a speci®c lysine residue of the intended E2 component (Perham, 1991(Perham, , 2000de Kok et al, 1998).…”

“…In addition, residues in the surface loop connecting b-strands 1 and 2 (Ile10, Gly11, Gly12 and Glu14) also underwent small yet signi®cant changes in chemical shift. This is not entirely surprising, as the surface loop is close in space to the lipoyl-lysine b-turn and has been implicated as a contact region with Elp in previous studies (Wallis et al, 1996;Jones et al, 2000b). The chemical shifts of residues in and surrounding b-strand 7 (Gly62, Lys64 and Gly68) were also slightly perturbed.…”

“…In this instance, our results indicate that the lipoyl-lysine and adjacent residues in the lipoyl domain are consistently involved in the interaction with E1p. The prominent surface loop linking b-strands 1 and 2 also seems to have a role to play, as it does in other complexes (Wallis et al, 1996;Berg et al, 1998;Jones et al, 2000b). However, in the E. coli E2plip apo domain, only Gly11 in the loop undergoes what was classed as a signi®cant change in chemical shift, and only the lipoyl-lysine region undergoes a T 2 change (Figure 1).…”

“…The addition of 2-oxoglutarate, the natural substrate for E1o, caused a slight change in chemical shift for three residues (Gly12, Glu16, Ala29), but the changes were much smaller than those observed in the presence of Elp and pyruvate (Figure 3) and the multiple resonances for speci®c residues ( Figure 2) were not observed. Thus, the presence of 2-oxoglutarate may promote an interaction of E2plip apo with E1o, but any such interaction is very weak and is certainly non-productive catalytically (Graham et al, 1989;Jones et al, 2000b).…”

Identification and Solution Structures of a Single Domain Biotin/Lipoyl Attachment Protein from Bacillus subtilis

“…Lipoic acid can serve as cofactor in a variety of proteins, such as E2 acyltransferases (E2p) in the pyruvate dehydrogenase complex and H-protein of the glycine cleavage system (4,5). It is also covalently bound via an amide linkage to a lysine group.…”